Multiplexed methylation detection methods

ABSTRACT

The present invention is directed to sensitive and accurate multiplexed assays for target analyte detection and detection of methylation in nucleic acid samples.

This application is a national phase under 35 U.S.C. §371 ofPCT/US2003/038582, filed Dec. 3, 2003, which is a continuation-in-partof U.S. application Ser. No. 10/309,803, filed Dec. 3, 2002, now U.S.Pat. No. 7,611,869, issued Nov. 3, 2009, both of which are incorporatedby reference in their entirety.

This invention was made in part with U.S. Government support under GrantNo. CA97851 awarded by the National Institutes of Health. The U.S.Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention is directed to sensitive and accurate multiplexedassays for target analyte detection.

BACKGROUND OF THE INVENTION

The detection of various target analytes or molecules is an importanttool for a variety of application including diagnostic medicine,molecular biology research and detection of contaminants, to name a few.While method of detecting different analytes has evolved, the ability todetect numerous target analytes simultaneously has proven difficult.Detection of multiple proteins, for example has been limited toconventional electrophoresis assays or immunoassays. There has not beena significant multiplexed protein detection assay or method.

The detection of specific nucleic acids is an important tool fordiagnostic medicine and molecular biology research. Gene probe assayscurrently play roles in identifying infectious organisms such asbacteria and viruses, in probing the expression of normal and mutantgenes and identifying mutant genes such as oncogenes, in typing tissuefor compatibility preceding tissue transplantation, in matching tissueor blood samples for forensic medicine, and for exploring homology amonggenes from different species.

Ideally, a gene probe assay should be sensitive, specific and easilyautomatable (for a review, see Nickerson, Current Opinion inBiotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. lowdetection limits) has been greatly alleviated by the development of thepolymerase chain reaction (PCR) and other amplification technologieswhich allow researchers to amplify exponentially a specific nucleic acidsequence before analysis (for a review, see Abramson et al., CurrentOpinion in Biotechnology, 4:41-47 (1993)).

Specificity, in contrast, remains a problem in many currently availablegene probe assays. The extent of molecular complementarity betweenprobe, and target defines the specificity of the interaction. Variationsin the concentrations of probes, of targets and of salts in thehybridization medium, in the reaction temperature, and in the length ofthe probe may alter or influence the specificity of the probe/targetinteraction.

It may be possible under some circumstances to distinguish targets withperfect complementarity from targets with mismatches, although this isgenerally very difficult using traditional technology, since smallvariations in the reaction conditions will alter the hybridization. Newexperimental techniques for mismatch detection with standard probesinclude DNA ligation assays where single point mismatches preventligation and probe digestion assays in which mismatches create sites forprobe cleavage.

Recent focus has been on the analysis of the relationship betweengenetic variation and phenotype by making use of polymorphic DNAmarkers. Previous work utilized short tandem repeats (STRs) aspolymorphic positional markers; however, recent focus is on the use ofsingle nucleotide polymorphisms (SNPs), which occur at an averagefrequency of more than 1 per kilobase in human genomic DNA. Some SNPs,particularly those in and around coding sequences, are likely to be thedirect cause of therapeutically relevant phenotypic variants and/ordisease predisposition. There are a number of well known polymorphismsthat cause clinically important phenotypes; for example, the apoE2/3/4variants are associated with different relative risk of Alzheimer's andother diseases (see Corder et al., Science 261:921-923 (1993). MultiplexPCR amplification of SNP loci with subsequent hybridization tooligonucleotide arrays has been shown to be an accurate and reliablemethod of simultaneously genotyping at least hundreds of SNPs; see Wanget al., Science, 280:1077 (1998); see also Schafer et al., NatureBiotechnology 16:33-39 (1998). However, in Wang et al. only 50% of 558SNPs were amplified successfully in a single multiplexed amplificationreaction. As such, there exists a need for methods that increase thefidelity and robustness of multiplexing assays.

Accordingly, highly multiplexed detection or genotyping of nucleic acidsequences is desirable to permit a new scale of genetic analysis.Simultaneously detecting many hundreds, to multiple thousands of nucleicacid sequences, will require methods which are sensitive and specificdespite high background complexity. In order for such reactions to beconducted at low cost to permit widespread use of such techniques,uniform sample preparation and reaction conditions must be applied,preferably in an automatable fashion. A variety of various nucleic acidreaction schemes, amplification techniques, and detection platforms havebeen used in the past toward this end goal, but none have been able torobustly achieve sensitive, accurate levels of multiplexing beyond a fewhundred loci.

In addition, DNA methylation is widespread and plays a critical role inthe regulation of gene expression in development, differentiation anddisease. Methylation in particular regions of genes, for example theirpromoter regions, can inhibit the expression of these genes (Baylin, S.B. and Herman, J. G. (2000) DNA hypermethylation in tumorigenesis:epigenetics joins genetics. Trends Genet, 16, 168-174.; Jones, P. A. andLaird, P. W. (1999) Cancer epigenetics comes of age. Nat Genet, 21,163-167.). Recent work has shown that the gene silencing effect ofmethylated regions is accomplished through the interaction ofmethylcytosine binding proteins with other structural compounds of thechromatin (Razin, A. (1998) CpG methylation, chromatin structure andgene silencing-a three-way connection. Embo J, 17, 4905-4908.; Yan, L.,Yang, X. and Davidson, N. E. (2001) Role of DNA methylation and histoneacetylation in steroid receptor expression in breast cancer. J MammaryGland Biol Neoplasia, 6, 183-192.), which, in turn, makes the DNAinaccessible to transcription factors through histone deacetylation andchromatin structure changes (Bestor, T. H. (1998) Gene silencing.Methylation meets acetylation. Nature, 393, 311-312.). Genomicimprinting in which imprinted genes are preferentially expressed fromeither the maternal or paternal allele also involves DNA methylation.Deregulation of imprinting has been implicated in several developmentaldisorders (Kumar, A. (2000) Rett and ICF syndromes: methylation movesinto medicine. J Biosci, 25, 213-214.; Sasaki, H., Allen, N. D. andSurani, M. A. (1993) DNA methylation and genomic imprinting in mammals.Exs, 64, 469-486.; Zhong, N., Ju, W., Curley, D., Wang, D., Pietrofesa,J., Wu, G., Shen, Y., Pang, C., Poon, P., Liu, X., Gou, S., Kajanoja,E., Ryynanen, M., Dobkin, C. and Brown, W. T. (1996) A survey of FRAXEallele sizes in three populations. Am J Med Genet, 64, 415-419.).

In vertebrates, the DNA methylation pattern is established early inembryonic development and in general the distribution of5-methylcytosine (5 mC) along the chromosome is maintained during thelife span of the organism (Razin, A. and Cedar, H. (1993) DNAmethylation and embryogenesis. Exs, 64, 343-357.; Reik, W., Dean, W. andWalter, J. (2001) Epigenetic reprogramming in mammalian development.Science, 293, 1089-1093.). Stable transcriptional silencing is criticalfor normal development, and is associated with several epigeneticmodifications. If methylation patterns are not properly established ormaintained, various disorders like mental retardation, immune deficiencyand sporadic or inherited cancers may follow. The study of methylationis particularly pertinent to cancer research as molecular alterationsduring malignancy may result from a local hypermethylation of tumorsuppressor genes, along with a genome wide demethylation (Schulz, W. A.(1998) DNA methylation in urological malignancies (review). Int J Oncol,13, 151-167.).

The initiation and the maintenance of the inactive X-chromosome infemale eutherians were found to depend on methylation (Goto, T. andMonk, M. (1998) Regulation of X-chromosome inactivation in developmentin mice and humans. Microbiol Mol Biol Rev, 62, 362-378.). Rett syndrome(RTT) is an X-linked dominant disease caused by mutation of MeCP2 gene,which is further complicated by X-chromosome inactivation (XCI) pattern.The current model predicts that MeCP2 represses transcription by bindingmethylated CpG residues and mediating chromatin remodeling (Dragich, J.,Houwink-Manville, I. and Schanen, C. (2000) Rett syndrome: a surprisingresult of mutation in MECP2. Hum Mol Genet, 9, 2365-2375.).

Finally, it has become a major challenge in epidemiological genetics torelate a biological function (e.g. a disease) not only to the genotypesof specific genes but also to the potential differential expressionlevels of each allele of the genes. DNA methylation data can providevaluable information, in addition to the genotype. While it is difficultto obtain the allele-specific methylation information, one object of theinvention is to provide methods to determine this information, e.g. if0, or 1 or 2 chromosomes are methylated at particular genomic locations.

In addition, the identification, classification and prognosticevaluation of tumors has until now depended on histopathologicalcriteria. The purpose of a classification scheme is to identifysubgroups of tumors with related properties, which can be furtherstudied and compared with each other. Such classification has been anessential first step in identifying the causes of various types ofcancer and in predicting their clinical behavior. However, molecular andbiochemical characteristics are not revealed by these approaches.Therefore, the current classification of tumors, although useful, isinsufficiently sensitive for prognostic assessment of individualpatients (especially for early diagnosis) and for probing the underlyingmechanisms involved. An integration of a broad range of information fromgenetic, biochemical and morphological approaches is needed.

The feasibility of molecular classification and prediction of cancershas been demonstrated using the method of monitoring overall geneexpression (Golub, T. R., Slonim, D. K., Tamayo, P., Huard, C.,Gaasenbeek, M., Mesirov, J. P., Coller, H., Loh, M. L., Downing, J. R.,Caligiuri, M. A., Bloomfield, C. D. and Lander, E. S. (1999) Molecularclassification of cancer: class discovery and class prediction by geneexpression monitoring. Science, 286, 531-537.). A mathematical model canbe developed to predict the disease type without prior pathologicaldiagnosis. However, it is rather difficult to produce reproducible andaccurate RNA-based gene expression profiling data under differentexperimental settings (Lockhart, D. J. and Winzeler, E. A. (2000)Genomics, gene expression and DNA arrays. Nature, 405, 827-836.).Furthermore, it is hard to compare the gene expression data generatedfrom different laboratories using different technology platforms andassay conditions (Roth, F. P. (2001) Bringing out the best features ofexpression data. Genome Res, 11, 1801-1802.). In addition, there, isscarce availability of reliable patient RNA samples.

DNA methylation pattern changes at certain genes often alter theirexpression, which could lead to cancer metastasis, for example. Thus, inone object of the invention a detailed study of methylation pattern inselected, staged tumor samples compared to matched normal tissues fromthe same patient offers a novel approach to identify unique molecularmarkers for cancer classification. Monitoring global changes inmethylation pattern has been applied to molecular classification inbreast cancer (Huang, T. H., Perry, M. R. and Laux, D. E. (1999)Methylation profiling of CpG islands in human breast cancer cells. HumMol Genet, 8, 459-470.). In addition, many studies have identified a fewspecific methylation patterns in tumor suppressor genes (for example,p16, a cyclin-dependent kinase inhibitor) in certain human cancer types(Herman, J. G., Merlo, A., Mao, L., Lapidus, R. G., Issa, J. P.,Davidson, N. E., Sidransky, D. and Baylin, S. B. (1995) Inactivation ofthe CDKN2/p16/MTS1 gene is frequently associated with aberrant DNAmethylation in all common human cancers. Cancer Res, 55, 4525-4530.;Otterson, G. A., Khleif, S. N., Chen, W., Coxon, A. B. and Kaye, F. J.(1995) CDKN2 gene silencing in lung cancer by DNA hypermethylation andkinetics of p16INK4 protein induction by 5-aza 2′deoxycytidine.Oncogene, 11, 1211-1216.).

RLGS profiling of methylation pattern of 1184 CpG islands in 98 primaryhuman tumors revealed that the total number of methylated sites isvariable between and in some cases within different tumor types,suggesting there may be methylation subtypes within tumors havingsimilar histology (Costello, J. F., Fruhwald, M. C., Smiraglia, D. J.,Rush, L. J., Robertson, G. P., Gao, X., Wright, F. A., Feramisco, J. D.,Peltomali, P., Lang, J. C., Schuller, D. E., Yu, L., Bloomfield, C. D.,Caligiuri, M. A., Yates, A., Nishikawa, R., Su Huang, H., Petrelli, N.J., Zhang, X., O'Dorisio, M. S., Held, W. A., Cavenee, W. K. and Plass,C. (2000) Aberrant CpG-island methylation has non-random andtumour-type-specific patterns. Nat Genet, 24, 132-138.). Aberrantmethylation of a proportion of these genes correlates with loss of geneexpression. Based on these observations, in one object of the inventionthe methylation pattern of a sizable group of tumor suppressor genes orother cancer-related genes will be used to classify and predictdifferent kinds of cancer, or the same type of cancer in differentstages.

Since methylation detection uses genomic DNA, but not the RNA, it offersadvantages in both the availability of the source materials and ease ofperforming the assays. Thus, the methylation assay will be complementaryto those based on RNA-based gene expression profiling. It is alsopossible that the use of different assays in combination may be moreaccurate and robust for disease classification and prediction.

Thus, methylation is involved in gene regulation. Altered methylationpatterns have been associated with various types of diseases includingcancers.

Accordingly, it is an object of the invention to provide methods forhigh-throughput genome-wide detection of genomic amplifications,deletions or methylation. For methylation, previously methods werelimited to detection of whether either one of the two chromosomes at alocus were methylated. However, it was not possible to determine if themethylation occurs on one or both chromosomes. Accordingly, the presentinvention provides a method for determining if zero, one or bothchromosomes are methylated at a locus.

Accordingly, it is an object of the invention to provide a verysensitive and accurate multiplexed approach for nucleic acid detectionand detection of methylation with uniform sample preparation andreaction conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a flow chart for array based detection of geneexpression.

FIG. 2 depicts a flow chart for array-based detection of RNA AlternativeSplicing.

FIG. 3 depicts a flow chart for genome-wide expression profiling usingoligonucleotide-ligation strategy.

FIG. 4 depicts a flow chart for genome-wide RNA alternative splicingmonitoring using oligonucleotide-ligation strategy.

FIG. 5 depicts a flow chart for direct genotyping using a whole-genomeoligonucleotide-ligation strategy.

FIG. 6 depicts a flow chart for whole-genome oligonucleotide-ligationstrategy.

FIG. 7 depicts a preferred embodiment of the invention utilizing apoly(A)-poly(T) capture to remove unhybridized probes and targets.Target sequence 5 comprising a poly(A) sequence 6 is hybridized totarget probe 115 comprising a target specific sequence 70, an adaptersequence 20, an upstream universal priming site 25, and a downstreamuniversal priming site 26. The resulting hybridization complex iscontacted with a bead 51 comprising a linker 55 and a poly(T) captureprobe 61.

FIG. 8 depicts a preferred embodiment of removing non-hybridized targetprobes, utilizing an OLA format. Target 5 is hybridized to a firstligation probe 100 comprising a first target specific sequence 15,detection position 10, an adapter sequence 20, an upstream universalpriming site 25, and an optional label 30, and a second ligation probe110 comprising a second target specific sequence 16, a downstreamuniversal priming site 26, and a nuclease inhibitor 35. After ligation,denaturation of the hybridization complex and addition of anexonuclease, the ligated target probe 115 and the second ligation probe110 is all that is left. The addition of this to an array (in thisembodiment, a bead array comprising substrate 40, bead 50 with linker 55and capture probe 60 that is substantially complementary to the adaptersequence 20), followed by washing away of the second ligation probe 110results in a detectable complex.

FIG. 9 depicts a preferred rolling circle embodiment utilizing twoligation probes. Target 5 is hybridized to a first ligation probe 100comprising a first target specific sequence 15, detection position 10,an adapter sequence 20, an upstream universal priming site 25, anadapter sequence 20 and a RCA primer sequence 120, and a second ligationprobe 110 comprising a second target specific sequence 16 and adownstream universal priming site 26. Following ligation, an RCAsequence 130 is added, comprising a first universal primer 27 and asecond universal primer 28. The priming sites hybridize to the primersand ligation occurs, forming a circular probe. The RCA sequence 130serves as the RCA primer for subsequent amplification. An optionalrestriction endonuclease site is not shown.

FIG. 10 depicts preferred a rolling circle embodiment utilizing a singletarget probe. Target 5 is hybridized to a target probe 115 comprising afirst target specific sequence 15, detection position 10, an adaptersequence 20, an upstream universal priming site 25, a RCA priming site140, optional label sequence 150 and a second target specific sequence16. Following ligation, denaturation, and the addition of the RCA primerand extension by a polymerase, amplicons are generated. An optionalrestriction endonuclease site is not shown.

FIG. 11 depicts two configurations of probes for multiplex detection ofanalytes. A depicts a probe containing an adapter 20, an upstreampriming site 25 and a target-specific portion, i.e. bioactive agent 160bound to a target analyte 7. B depicts a probe containing an adapter 20,an upstream universal priming site 25, a downstream universal primingsite 26 and a target-specific portion, i.e. bioactive agent 160 bound toa target analyte 7.

FIG. 12 depicts a preferred method for multiplex detection of analytes.Probes containing universal priming sequence 25 and adapters thatidentify the target analyte to be detected 21, 22 and 23, and targetspecific portions, i.e. bioactive agents 161, 162 and 163 are contactedwith target analytes 201 and 202. Probes to which target analytes bindare contacted with universal primers 210 and amplification reactionmixture. Amplicons are detected and serve as an indication of thepresence of the target analyte.

FIG. 13 In vitro controls for methylation profiling in the presence ofcomplex genome with a readout on fiber optic arrays. a). Panels A and Bshow that the signal detected by bead arrays from the plasmid-specificprimers (red bars, 1-5 in panel A) completely disappears after Hpa IIdigest of unmethylated DNA (1-5 in panel B). Plasmid primers do notcross-react with genomic DNA. Primer 1 used as a negative control has nohomology site on the pUC19 plasmid and shows no signal. b). Panels C andD demonstrate that in vitro methylated plasmid DNA is completelyresistant to Hpa II digest. Signals from plasmid primers (red bars, 1-5)and genomic DNA primers (yellow bars, 16-31) are specific. Genesrepresented in columns 16, 17, 20, 24 and 28 have a Hpa II site at ornear the primer annealing site. Note that signals in the columns 16 and17 on the panel C disappear completely on the panel D, which mayindicate unmethylated status of the targeted loci. The signals from theplasmid primers remained unchanged and confirm that pUC19 was completelymethylated. c). Panels E and F show that addition of genotyping probes(blue bars, 6-15) in combination with plasmid primers (red bars, 1-5)designed for non-methylated DNA can be used to monitor the quality ofhybridization process and DNA treatment.

SUMMARY OF THE INVENTION

In accordance with the embodiments outlined above, the present inventionpermits highly multiplexed detection of target analytes. The methodincludes contacting target analytes with a composition comprising anamplification enzyme and first and second target probes. The first andsecond target probes comprising a first and second bioactive agent,respectively, that specifically bind to the first and second targetmolecules. The probes also comprise a first and second adapter sequence,respectively, such that the first adapter sequence identifies the firsttarget molecule and the second adapter sequence identifies the secondtarget molecule, and at least a first and second upstream universalpriming sequence, respectively. The first and second adapter sequences,wherein no ligation is performed, to form first and second amplicons,respectively, and detecting the first and second amplicons, whereby thefirst and second target molecules, respectively, are detected.

In addition, the invention provides a method for multiplex detection ofa plurality of target molecules comprising contacting a plurality oftarget molecules with a composition comprising an amplification enzymeand a plurality of target probes, each comprising a bioactive agent,wherein the bioactive agent binds to discrete target molecules anadapter sequence that identifies the discrete target molecule that bindsthe bioactive agent and at least a first upstream universal primer,amplifying the adapter sequences, wherein no ligation is performed, toform a plurality of amplicons, and detecting the plurality of amplicons,whereby the plurality of target molecules, are detected.

In addition, present invention permits highly multiplexed nucleic aciddetection reactions under uniform sample preparation and reactionconditions. That is, preferably the method includes multiplexing fromhundreds to thousands of assays simultaneously, more preferably up totens of thousands of assays simultaneously, most preferably up tomillions of assays. The inventive method preferably includes 1)immobilizing the sample nucleic acids to be interrogated (in a preferredembodiment, genomic DNA) on a capture surface, such as a solid phase (ina preferred embodiment, immobilizing the genomic DNA on beads); 2)simultaneously conducting at least a first step of a nucleic aciddetection reaction with the captured nucleic acids (in the preferredembodiment, the nucleic acid detection reaction comprises two phases:the first phase involves the exposure of the sample nucleic acids to aset of sequence-specific probe(s), the second phase involves anenzymatic step to assure specificity of the nucleic acid detectionreaction. The probes used include at least one appropriate universalamplification priming site); 3) a stringent wash step to reduce thecomplexity of the multiplexed probe mixture by washing away unhybridizedprobes; 4) optionally conducting the second phase of the nucleic aciddetection reaction step of 2) above (in the case of for examplecompetitive hybridization as the nucleic acid detection reaction, nosecond phase is required); 5) releasing the probes from the samplenucleic acid; 6) amplification of the released probes (exponential orlinear amplification schemes such as PCR, or Invader™, ESPIA (see WO01/20035, which is expressly incorporated herein by reference), T7amplification or the novel amplification method disclosed in Applicationpatent application filed Jul. 12, 2001, entitled METHODS OF MULTIPLEXINGAMPLIFICATION AND GENOTYPING REACTIONS (no serial number received))using the universal amplification priming site(s) on the probes; and 6)detection and readout of the amplified signals on any detection platform(in a preferred embodiment, the randomly assembled BeadArray™ technologyplatform).

In addition the invention provides a method for multiplex detection ofmethylation of target nucleic acids comprising providing a firstpopulation of target nucleic acids labeled with a purification tag,cleaving the first population of target nucleic acids with an enzyme,whereby the enzyme discriminately cleaves at methylation targetsequences forming a second population of cleaved target sequences,immobilizing the first and second populations by the purification tagand detecting the presence of the first population comprisingnon-cleaved target nucleic acid whereby the presence of the firstpopulation comprising non-cleaved target nucleic acid indicates thepresence of methylated target nucleic acids.

The invention also provides a method of detecting methylation contactinga sample of target nucleic acids with bisulfite, whereby non-methylatedcytosine is converted to uracil, and methylated cytosine is notconverted to uracil; contacting said treated target nucleic acids with afirst probe that hybridizes with a methylated target in said firstpopulation of target nucleic acid and a second probe that hybridizeswith a non-methylated target in said second population of target nucleicacid, forming first and second hybridization complexes, respectively;contacting said first and second hybridization complexes with an enzymethat modifies said first and second probes forming first and secondmodified probes; and detecting said first and second modified probes todetermine the presence of methylation in said target nucleic acid.

In addition the invention provides a method of detecting methylationcomprising contacting a sample of target nucleic acids with bisulfite,whereby non-methylated cytosine is converted to uracil forming a firstpopulation of treated target nucleic acids, and methylated cytosine isnot converted to uracil forming a second population of treated targetnucleic acids, contacting the first and second populations of treatedtarget nucleic acids with a first probe that hybridizes with a firsttarget in the first population of target nucleic acid and a second probethat hybridizes with a target in the second population of target nucleicacid, forming first and second hybridization complexes, respectively,contacting the first and second hybridization complexes with an enzymethat modifies the first and second probes forming first and secondmodified probes, and detecting the first and second modified probes todetermine the presence of methylation in the target nucleic acid.

The invention also provides methods for detecting methylation that allowfor whole genome amplification after bisulfite conversion in a mannerthat takes advantage of the unique sequence feature introduced intogenomic DNA sequences by sodium bisulfite treatment, which is thedeamination of cytosine, but not 5-methylcytosine, to uracil. In oneembodiment, the invention provides a method of detecting methylationfollowing bisulfite conversion of target nucleic acids, said methodcomprising providing target nucleic acids that have undergone bisulfiteconversion, contacting said target nucleic acids with two sets ofprimers, wherein a first set of said two sets of primers has a higheraffinity for an original bisulfite converted strand of said targetnucleic acid compared to a corresponding complementary strand that hasnot undergone bisulfite conversion and wherein a second set of said twosets of primers has a higher affinity for said correspondingcomplementary strand compared to said original bisulfite convertedstrand of said target nucleic acid, wherein said contacting is doneunder conditions that allow the primers to hybridize to a complimentarynucleic acid sequence, and amplifying said bisulfite converted strand ofsaid target nucleic acid and said corresponding complementary strand. Ina related embodiment, an initial contacting step is performed thatfurther comprises addition of a set of poly-A primers.

The invention also provides a method for detecting methylation followingbisulfite conversion of target nucleic acids, the method comprisingproviding target nucleic acids that have undergone bisulfite conversion,contacting the target nucleic acids with primer sequences comprisingpoly-Adenines to synthesize a population of first strand products,modifying said population of first strand products to add poly-Thyminesequences; contacting said modified first strand products withpoly-Adenine primers under conditions that allow the primers tohybridize to a complimentary nucleic acid sequence; and amplifying saidmodified first strand products. In particular embodiments, modifying thepopulation of first strand products can be carried out by contacting thepopulation of first strand products with Thymine in the presence of anenzyme that allows for adding poly-Thymine sequences to the first strandproducts. The addition of poly-Thymine sequences to a population offirst strand products can be accomplished using any of a variety ofmethods known in the art including, for example, use of a terminaldeoxyribonucleotide transferase enzyme in the presence of thyminetriphosphate. Other methods include, without limitation, chemicalsynthesis or nucleotide extension methods in which the first strandproducts are extended in the presence of an annealed template having apoly adenine sequence.

Also provided is a method for generating a calibration curve for thequantitative methylation measurement of an unknown sample, said methodcomprising providing and amplifying a first reference genomic DNA, forexample, at least one hundred fold, one thousand fold, ten thousand foldor more, wherein the ratio of methylated to unmethylated sequences isdecreased sufficiently to prepare a virtually unmethylated templatepopulation; obtaining a methylated template population comprisingnucleotide sequences corresponding to the nucleotide sequences of saidreference genomic DNA; and separately mixing fixed amounts of saidvirtually unmethylated template population with fixed amounts of saidmethylated template population at various distinct ratios to create aseries of mixtures that represents a methylation gradient, wherein acalibration curve is generated for quantitative methylationmeasurements. In this embodiment, the invention also provides a methodfor producing a calibration reagent for quantitative methylationmeasurement of an unknown sample of the reference organism.

The invention also provides a calibration curve and a calibrationreagent prepared by the method described above that allows forquantitative calibration of any genomic DNA methylation measurement,regardless of the methylation detection system utilized. The inventionalso provides a calibration curve comprising a first axis indicating theamount of unmethylated template population compared to methylatedtemplate population and a second axis indicating a detected methylationsignal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the multiplex preparation anddetection of methylated target nucleic acids. In general, the inventioninvolves the use of methylation selective modification of target nucleicacids and detection of the modified target nucleic acids. In oneembodiment the methylation selective modification involves cleavingtarget nucleic acids with methylation selective enzymes and detection ofthe cleaved or uncleaved nucleic acids with probes. That is, in apreferred embodiment the method involves providing a first population oftarget nucleic acids as described herein and cleaving or shearing thefirst population so as to reduce the size of each target. The targetnucleic acids can be any region including but not limited tonon-polymorphic regions. The sized target nucleic acids are then labeledwith a purification tag as described herein. The labeled target nucleicacids are cleaved with an enzyme that discriminately cleaves atmethylated sites, that is the enzyme either selectively cleaves or doesnot cleave at a site that is methylated. Therefore, the term “methylatedtarget sequence” as used in reference to an enzyme that discriminatelycleaves at the site of the target sequence refers to target sequencesfor methylation, regardless of methylation status, and areinterchangeably referred to as “methylation target sequences.”Typically, the discrimination is based on methylation blockingrestriction such that methylation target sequences in non-methylatedstate are discriminately cleaved by the enzyme. Generally and preferablythe enzyme has sequence specificity in addition to methylationsensitivity, such as Hpa II. This cleaved and labeled mixture is thenimmobilized to a solid support. Generally this is accomplished by thepurification tag. Detection of the methylated target sequence is thenperformed, using any variety of assays. Generally, these assays rely onprimers or probes that span the junction site.

In addition, the results obtained from different gDNAs treated with orwithout methylation selective enzymes can be compared to deduce thegenomic methylation pattern.

In another embodiment the method involves the use of bisulfite. That is,in an alternative method for detecting methylation, the fact thatnon-methylated cytosines are converted to uracils when treated withbisulfite is exploited. The hybridization properties of uracil aresimilar to that of thymine. Thus, when the sample DNA is treated withbisulfite, non-methylated cytosine hybridizes like thymine, whilemethylated cytosine will hybridize like cytosine. This difference inhybridization properties can be detected by using appropriate targetprobes. That is, the methylated or non-methylated cytosine site can betreated as a C/T polymorphic site and detected by any of the assays asdescribed herein. The resulting modified target probes are detected byany of the detection methods as described herein.

In addition the invention provides a method for detecting methylationfollowing bisulfite conversion of target nucleic acids, the methodcomprising providing target nucleic acids that have undergone bisulfiteconversion, contacting the target nucleic acids with two sets ofprimers, wherein a first set of the two sets of primers has a higheraffinity for an original bisulfite converted strand of the targetnucleic acid compared to a corresponding complementary strand that hasnot undergone bisulfite conversion and wherein a second set first set ofthe two sets of primers has a higher affinity for the correspondingcomplementary strand compared to the original bisulfite converted strandof the target nucleic acid, wherein the contacting is done underconditions that allow the primers to hybridize to a complimentarynucleic acid sequence, and amplifying the bisulfite converted strand ofthe target nucleic acid and the corresponding complementary strand. In arelated embodiment, an initial contacting step is performed that furthercomprises addition of a set of poly-A primers. An exemplary set ofprimers having higher affinity for an original bisulfite convertedstrand of a target nucleic acid compared to a correspondingcomplementary strand that has not undergone bisulfite conversion includea set of primers that contains combinations of A, T and C nucleotidesbut not G as described in Example 3.

The invention also provides a method for detecting methylation followingbisulfite conversion of target nucleic acids, the method comprisingproviding target nucleic acids that have undergone bisulfite conversion,contacting the target nucleic acids with primer sequences comprisingpoly-Adenines to synthesize a population of first strand products,modifying said population of first strand products to add poly-Thyminesequences; contacting said modified first strand products withpoly-Adenine primers under conditions that allow the primers tohybridize to a complimentary nucleic acid sequence; and amplifying saidmodified first strand products. In particular embodiments, modifying thepopulation of first strand products can be carried out by contacting thepopulation of first strand products with Thymine in the presence of anenzyme that allows for adding poly-Thymine sequences to the first strandproducts.

Poly-Thymine tracts can be added to a population of first strandproducts using any of a variety of methods known in the art including,for example, use of a terminal deoxyribonucleotide transferase enzyme inthe presence of thymine triphosphate. Other methods include, withoutlimitation, chemical synthesis or nucleotide extension methods in whichthe first strand products are extended in the presence of an annealedtemplate having a poly adenine sequence.

The invention methods for detecting methylation allow for whole genomeamplification after bisulfite conversion in a manner that takesadvantage of the unique sequence feature introduced into genomic DNAsequences by sodium bisulfite treatment, which is the deamination ofcytosine, but not 5-methylcytosine, to uracil.

Thus, the invention also involves the use of probes that comprise anumber of components. First of all, the probes comprise a bioactiveagent (e.g. one of a binding partner pair) that will bind to all or aportion of the target nucleic acid. This bioactive agent preferablycomprises nucleic acid, because the target analyte is a target nucleicacid sequence. The probes further comprise at least one adapter nucleicacid sequence that uniquely identifies the target nucleic acid. That is,there is a unique adapter sequence/target nucleic acid pair for eachunique target nucleic acid, although in some cases, adapter sequencesmay be reused.

In addition, the probes also comprise at least one universal nucleicacid priming sequence that will allow the amplification of the adaptersequence. In some cases, one universal priming sequence can be used, forexample when the priming sequence comprises an RNA polymerase primingsequence such as a T7 site. Alternatively, two universal primingsequences can be used, such as standard PCR priming sequences, as longas they flank the adapter sequence, e.g. one priming sequence is 5′ tothe adapter sequence and one is 3′. Once the probes have been added tothe target nucleic acids to form assay complexes (sometimes referred toherein as hybridization complexes) generally the unhybridized probes arewashed away, using a variety of techniques as outlined herein.

Amplification proceeds in a number of ways. In general, when an RNApolymerase priming sequence is used such as a T7 site, the RNApolymerase is added and copies of the adapter sequence are generated.Alternatively, when the amplification reaction is PCR, two primers areadded, each of which is substantially complementary (and preferablyperfectly complementary) to one of the universal priming sequences orits complement. Again, as outlined more fully below, there may be morethan one set of universal priming sequences/primers used in a givenreaction. In addition, as will be appreciated by those in the art, anumber of other amplification reactions can be done, as outlined below.

In an alternative embodiment, the “Universal” primer sequences aredesigned not to solely serve as PCR primers, but also as a promotersequence for RNA Polymerase. Thus, the annealed (and/or ligated) targetprobes can be amplified not only by general PCR, but can also beamplified by in vitro transcription (IVT). The linear amplificationproduced by IVT should be better at maintaining the relative amounts ofthe different sequences in the initial template population than theexponential amplification of PCR.

The resulting amplicons can be detected in a wide variety of ways,including the use of biochips (e.g. solid support arrays, including bothordered and random arrays, as outlined herein) liquid arrays, capillaryelectrophoresis, mass spectroscopy analysis, etc., in a variety offormats, including sandwich assays, as is further described herein.

In some cases, one or more of the target analytes or probes may beattached to a solid support. For example, the target analytes (forexample, genomic DNA sequences) can be attached to beads in a variety ofways. The probe pool is added to form assay complexes (sometimesreferred to herein as hybridization complexes when the target analytesare nucleic acids) and unhybridized probes are washed away. The probesare denatured off the target analytes, and then amplified as outlinedherein.

Alternatively, solution phase assays may be done, followed by eitherliquid or solid array detection.

Accordingly, the present invention relates to the multiplexamplification and detection of methylated target nucleic acids in asample. As used herein, the phrase “multiplex” or grammaticalequivalents refers to the detection, analysis or amplification of morethan one target nucleic acid of interest. In a one embodiment, multiplexrefers to at least 100 different target nucleic acids while at least 500different target nucleic acids is preferred. More preferred is at least1000, with more than 5000 particularly preferred and more than 10,000most preferred. Detection is performed on a variety of platforms. In apreferred embodiment the invention is utilized with adapter sequencesthat identify the target molecule.

In addition, the present invention provides compositions and methods fordetecting methylated target nucleic acids including detecting andquantitating specific, methylated target nucleic acid sequences in asample. As will be appreciated by those in the art, the sample solutionmay comprise any number of things, including, but not limited to, bodilyfluids (including, but not limited to, blood, urine, serum, lymph,saliva, anal and vaginal secretions, perspiration and semen, ofvirtually any organism, with mammalian samples being preferred and humansamples being particularly preferred). The sample may compriseindividual cells, including primary cells (including bacteria), and celllines, including, but not limited to, tumor cells of all types(particularly melanoma, myeloid leukemia, carcinomas of the lung,breast, ovaries, colon, kidney, prostate, pancreas and testes),cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-celland B cell), mast cells, eosinophils, vascular intimal cells,hepatocytes, leukocytes including mononuclear leukocytes, stem cellssuch as haemopoetic, neural, skin, lung, kidney, liver and myocyte stemcells, osteoclasts, chondrocytes and other connective tissue cells,keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes.Suitable cells also include known research cells, including, but notlimited to, Jurkat T cells, NIH3T3 cells, CHO, Cos, 923, HeLa, WI-38,Weri-1, MG-63, etc. See the ATCC cell line catalog, hereby expresslyincorporated by reference.

If required, the target analyte is prepared using known techniques. Forexample, the sample may be treated to lyse the cells, using known lysisbuffers, sonication, electroporation, etc., with purification andamplification as outlined below occurring as needed, as will beappreciated by those in the art. In addition, the reactions outlinedherein may be accomplished in a variety of ways, as will be appreciatedby those in the art. Components of the reaction may be addedsimultaneously, or sequentially, in any order, with preferredembodiments outlined below. In addition, the reaction may include avariety of other reagents which may be included in the assays. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc., which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target.

In addition, when nucleic acids are to be detected preferred methodsutilize cutting or shearing techniques to cut the nucleic acid samplecontaining the target sequence into a size that will facilitate handlingand hybridization to the target, particularly for genomic DNA samples.This may be accomplished by shearing the nucleic acid through mechanicalforces (e.g. sonication) or by cleaving the nucleic acid usingrestriction endonucleases.

In addition, in most embodiments, double stranded target nucleic acidsare denatured to render them single stranded so as to permithybridization of the primers and other probes of the invention. Apreferred embodiment utilizes a thermal step, generally by raising thetemperature of the reaction to about 95° C., although pH changes andother techniques may also be used.

In one preferred embodiment the target nucleic acids have been preparedas described below to detect the presence or absence of methylation atvarious loci.

In a preferred embodiment, the compositions and methods of the inventionare directed to the detection of target sequences. By “nucleic acid” or“oligonucleotide” or grammatical equivalents herein means at least twonucleotides covalently linked together. A nucleic acid of the presentinvention will generally contain phosphodiester bonds, although in somecases, as outlined below, particularly for use with probes or primers,nucleic acid analogs are included that may have alternate backbones,comprising, for example, phosphoramide (Beaucage et al., Tetrahedron49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem.35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977);Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem.Lett. 13:805-808 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); and Pauwels et al., Chemica Scripta 26:141 91986)),phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); andU.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem.Soc. 111:2321 (1989), O-methylphosphoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature380:207 (1996), all of which are incorporated by reference). Otheranalog nucleic acids include those with positive backbones (Denpcy etal., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of labels, or to increase the stability and half-life ofsuch molecules in physiological environments.

In a preferred embodiment, the nucleic acid preferably includes at leastone universal base. Universal bases are those that can substitute forany of the five natural bases, that is, universal bases will basepairwith all natural bases, preferably equally well. Suitable universalbases include, but are not limited to, inosine, hypoxanthine,5′nitroindole, acylic 5″nitroindole, 4′nitropyrazole, 4′nitroimidazoleand 3′nitropyrrole. See Loakes et al., Nucleic Acid Res. 22:4039 (1994);Van Aerschot et al., Nucleic Acid Res. 23:4363 (1995); Nichols et al.,Nature 369:492 (1994); Berstrom et al., Nucleic Acid Res. 25:1935(1997); Loakes et al., Nucleic Acid Res. 23:2361 (1995); Loakes et al.,J. Mol. Biol. 270:426 (1997); and Fotin et al., Nucleic Acid Res.26:1515 (1998); and references cited therein, all of which are expresslyincorporated by reference.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C. Thisallows for better detection of mismatches. Similarly, due to theirnon-ionic nature, hybridization of the bases attached to these backbonesis relatively insensitive to salt concentration.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. Thus, for example, when the target sequence is apolyadenylated mRNA, the hybridization complex comprising the targetprobe has a double stranded portion, where the target probe ishybridized, and one or more single stranded portions, including thepoly(A) portion. The nucleic acid may be DNA, both genomic and cDNA, RNAor a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. A preferred embodimentutilizes isocytosine and isoguanine in nucleic acids designed to becomplementary to other probes, rather than target sequences, as thisreduces non-specific hybridization, as is generally described in U.S.Pat. No. 5,681,702. As used herein, the term “nucleoside” includesnucleotides as well as nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occurring analog structures. Thusfor example the individual units of a peptide, nucleic acid, eachcontaining a base, are referred to herein as a nucleoside.

Preferably the target sequence is potentially a methylated targetsequence. Generally “methylated” includes any nucleotide that ismethylated. Frequently methylated refers to nucleic acids that include5-methylcytosine, but the term also can be used in the context of anenzyme that discriminately cleaves at the site of sequences that aretargets for methylation, referred to interchangeably as “methylatedtarget sequences” or “methylation target sequences.” The term “targetsequence” or “target nucleic acid” or grammatical equivalents hereinmeans a nucleic acid sequence on a single strand of nucleic acid. Thetarget sequence may be a portion of a gene, a regulatory sequence,genomic DNA (gDNA), cDNA, RNA including mRNA and rRNA, or others, withpotentially methylated genomic DNA being particular preferred in someembodiments. As described above, the term “methylated target sequence”when used in reference to an enzyme that discriminately cleaves at thesite of the target sequence refers to target sequences for methylation,regardless of methylation status, and are interchangeably referred to as“methylation target sequences.” Typically, the discrimination is basedon methylation blocking restriction such that methylation targetsequences in non-methylated state are discriminately cleaved by theenzyme.

As is outlined herein, the target sequence may be a target sequence froma sample, or a secondary target such as an amplicon, which is theproduct of an amplification reaction such as PCR or an RNA polymerasereaction, although generally the target sequence will be from a sample.

The target sequence may be any length, with the understanding thatlonger sequences are more specific. As will be appreciated by those inthe art, the complementary target sequence may take many forms. Forexample, it may be contained within a larger nucleic acid sequence, i.e.all or part of a gene or mRNA, a restriction fragment of a plasmid orgenomic DNA, among others. Particularly preferred target sequences inthe present invention include genomic DNA, polyadenylated mRNA, andalternatively spliced RNAs. As is outlined more fully below, probes aremade to hybridize to target sequences to determine the presence,absence, quantity or sequence of a target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart.

The target sequence may also be comprised of different target domains,that may be adjacent (i.e. contiguous) or separated. The terms “first”and “second” are not meant to confer an orientation of the sequenceswith respect to the 5′-3′ orientation of the target sequence. Forexample, assuming a 5′-3′ orientation of the complementary targetsequence, the first target domain may be located either 5′ to the seconddomain, or 3′ to the second domain. In addition, as will be appreciatedby those in the art, the probes on the surface of the array (e.g.attached to the microspheres) may be attached in either orientation,either such that they have a free 3′ end or a free 5′ end; in someembodiments, the probes can be attached at one or more internalpositions, or at both ends.

In a preferred embodiment the invention is directed to target sequencesthat comprise one or more positions for which sequence information isdesired, generally referred to herein as the “detection position” or“detection locus”. In a preferred embodiment, the detection position isa single nucleotide, generally a cytosine that may, at times, bemethylated, although in some embodiments, it may comprise either othersingle nucleotides or a plurality of nucleotides, either contiguous witheach other or separated by one or more nucleotides. By “plurality” asused herein is meant at least two. As used herein, the base of a probe(e.g. the target probe) which basepairs with a detection position base,in a hybrid is termed a “readout position” or an “interrogationposition”. Thus, the target sequence comprises a detection position andthe target probe comprises a readout position.

In a preferred embodiment, the use of competitive hybridization targetprobes is done to elucidate either the identity of the nucleotide(s) atthe detection position or the presence of a mismatch.

It should be noted in this context that “mismatch” is a relative termand meant to indicate a difference in the identity of a base at aparticular position, termed the “detection position” herein, between twosequences. In general, sequences that differ from wild type sequencesare referred to as mismatches. In the case of SNPs, what constitutes“wild type” may be difficult to determine as multiple alleles can berelatively frequently observed in the population, and thus “mismatch” inthis context requires the artificial adoption of one sequence as astandard. When determining methylation patterns, the sequence of thetarget prior to any modification as set forth herein constitutes “wildtype” while the sequence subsequent to any methylation selectivemodification constitutes a “mismatch”. Thus, for the purposes of thisinvention, sequences are referred to herein as “match” and “mismatch”.Thus, while the present invention may be used to detect substitutions,insertions or deletions as compared to a wild-type sequence, preferablythe invention is used to detect methylation of target nucleic acids.That is, all other parameters being equal, a perfectly complementaryreadout target probe (a “match probe”) will in general be more stableand have a slower off rate than a target probe comprising a mismatch (a“mismatch probe”) at any particular temperature.

In a preferred embodiment the target nucleic acids are modified in amethylation selective manner either prior to or after immobilization ofthe target nucleic acids as described below. That is, DNA methylationanalysis methods generally rely on a methylation-dependent modificationof the original genomic DNA before any amplification step. The methodsare outlined generally below.

Methylation-Specific Enzymes

In one embodiment a method of methylation detection assays includesdigesting genomic DNA with a methylation-sensitive restriction enzymefollowed by detection of the differentially cleaved DNA, e.g. bySouthern blot analysis (Issa, J. P., Ottaviano, Y. L., Celano, P.,Hamilton, S. R., Davidson, N. E. and Daylin, S. B. (1994) Methylation ofthe oestrogen receptor CpG island links ageing and neoplasia in humancolon. Nat Genet, 7, 536-540.; Taylor, J. M., Kay, P. H. and Spagnolo,D. V. (2001) The diagnositc significance of Myf-3 hypermethylation inmalignant lymphoproliferative disorders. Leukemia, 15, 583-589) or PCR(Singer-Sam, J., LeBon, J. M., Tanguay, R. L. and Riggs, A. D. (1990) Aquantitative HpaII-PCR assay to measure methylation of DNA from a smallnumber of cells. Nucleic Acids Res, 18, 687), or methods as describedbelow. In a preferred embodiment the methylation specific enzyme isHpaII which recognizes 5′-CCGG-3′. The digestion is blocked bymethylation at either C. Also, the methylation specific enzyme Msp Ifinds use in the invention.

Bisulfite DNA Sequencing

In a preferred embodiment, the method is based on the selectivedeamination of cytosine to uracil by treatment with bisulfite and thesequencing of subsequently generated PCR products. The method utilizesbisulfite-induced modification of genomic DNA, under conditions wherebycytosine is converted to uracil, but 5-methylcytosine remainsnon-reactive. The sequence under investigation is then analyzed by anyof the methods as described below including without limitation, beingamplified by PCR with two sets of strand-specific primers to yield apair of fragments, one from each strand, in which all uracil and thymineresidues have been amplified as thymine and only 5-methylcytosineresidues have been amplified as cytosine. The PCR products can bedetected as described below or sequenced directly to provide astrand-specific average sequence for the population of molecules or canbe cloned and sequenced to provide methylation maps of single DNAmolecules (Feil, R., Charlton, J., Bird, A. P., Walter, J. and Reik, W.(1994) Methylation analysis on individual chromosomes: improved protocolfor bisulphite genomic sequencing. Nucleic Acids Res, 22, 695-696;Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F.,Grigg, G. W., Molloy, P. L. and Paul, C. L. (1992) A genomic sequencingprotocol that yields a positive display of 5-methylcytosine residues inindividual DNA strands. Proc Natl Acad Sci USA, 89, 1827-1831). Exactmethylation maps of single DNA strands from individual genomic DNAmolecules can be established, where the position of each5-methylcytosine is given by a clear positive band on a sequencing gel.

Methylation-Specific PCR (MSP)

In an alternative embodiment the method includes an initial modificationof DNA by sodium bisulfite, and subsequent detection and amplificationwith primers specific for methylated versus unmethylated DNA (Herman, J.G., Graff, J. R., Myohanen, S., Nelkin, B. D. and Baylin, S. B. (1996)Methylation-specific PCR: a novel PCR assay for methylation status ofCpG islands. Proc Natl Acad Sci USA, 93, 9821-9826.) and as described inmore detail below. The method can rapidly assess the methylation statusof virtually any group of CpG sites within a CpG island, and does notrequire the use of methylation-sensitive restriction enzymes. MSPrequires only small quantities of DNA, is sensitive to 0.1% methylatedalleles of a given CpG island locus, and can be performed on DNAextracted from paraffin-embedded samples. Unmodified DNA or DNAincompletely reacted with bisulfite can be distinguished, since markedsequence differences exist between these DNAs. Simultaneous detection ofunmethylated and methylated products in a single sample allows asemi-quantitative assessment of allele types that approximates thequantitation determined by Southern analysis. The ability to validatethe amplified product by differential restriction patterns is anadditional advantage.

Methylation-Sensitive Single Nucleotide Primer Extension (MS-SnuPE)

In an alternative embodiment the method includes treating genomic DNAswith bisulfite and the target sequences are amplified with PCR primersspecific for the converted DNA. The resulting PCR products are then usedas a template for the MS-SnuPE reaction, in the presence of specificextension primers and dye-labeled or radioactive dCTP or dTTP. Theextension primers are designed in such that their 3′-landing sites arejust one base before the incorporation site designated for methylationanalysis. If the target site is methylated, a C will be incorporatedduring the primer extension, or a T will be incorporated if the targetsite is unmethylated. Quantitation of the relative C and T incorporationwill allow the determination of the methylation status of the targetsite. A complete bisulfite-mediated DNA conversion is important for anaccurate measurement (of methylation) with this approach. This methodfinds use in quantitation of methylation difference at specific CpGsites (Gonzalgo, M. L. and Jones, P. A. (1997) Rapid quantitation ofmethylation differences at specific sites using methylation-sensitivesingle nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res, 25,2529-2531; Kuppuswamy, M. N., Hoffmann, J. W., Kasper, C. K., Spitzer,S. G., Groce, S. L. and Bajaj, S. P. (1991) Single nucleotide primerextension to detect genetic diseases: experimental application tohemophilia B (factor IX) and cystic fibrosis genes. Proc Natl Acad SciUSA, 88, 1143-1147).

Other methods such as Restriction landmark genomic scanning (RLGS)(Akama, T. O., Okazaki, Y., Ito, M., Okuizumi, H., Konno, H., Muramatsu,M., Plass, C., Held, W. A. and Hayashizaki, Y. (1997) Restrictionlandmark genomic scanning (RLGS-M)-based genome-wide scanning of mouseliver tumors for alterations in DNA methylation status. Cancer Res, 57,3294-3299.; Kawai, J., Hirose, K., Fushiki, S., Hirotsune, S., Ozawa,N., Hara, A., Hayashizaki, Y. and Watanabe, S. (1994) Comparison of DNAmethylation patterns among mouse cell lines by restriction landmarkgenomic scanning. Mol Cell Biol, 14, 7421-7427) and differentialmethylation hybridization (DMH) (Huang, T. H., Perry, M. R. and Laux, D.E. (1999) Methylation profiling of CpG islands in human breast cancercells. Hum Mol Genet, 8, 459-470) also find use in the invention. Allreferences are expressly incorporated herein by reference.

In addition to the methods described above as useful to assess ormeasure the methylation status at a given site in a genomic DNA, forexample, bisulfite sequencing and methylation-specific PCR, theinvention provides a calibration curve for the quantitative methylationmeasurement of an unknown sample derived from a reference organism. Thecalibration curve of the invention can be prepared by providing andamplifying a first reference genomic DNA, for example, at least onehundred fold, one thousand fold, ten thousand fold, or more, wherein theratio of methylated to unmethylated sequences is decreased sufficientlyto prepare a virtually unmethylated template population, obtaining amethylated template population having nucleotide sequences correspondingto the nucleotide sequences of the reference genomic DNA, separatelymixing fixed amounts of the virtually unmethylated template populationwith fixed amounts of the methylated template population at variousdistinct ratios to create a series of mixtures that represents amethylation gradient, thereby generating a calibration reagent forquantitative methylation measurement of an unknown sample of thereference organism. The methylated template population can be obtainedby methylating the reference genomic DNA. If desired the methylatedtemplate population can be obtained by methylating a portion removedfrom the virtually unmethylated sample. Nucleic acid samples can bemethylated using an enzyme biological sample, or fraction thereof havingDNA methylation activity such as those described herein or known in theart.

The invention thus provides a calibration curve that allowsquantitatively calibrate any genomic DNA methylation measurement,regardless of the methylation detection system that is utilized Acalibration curve of the invention can be prepared by the methoddescribed above. The invention also provides a calibration curvecomprising a first axis indicating the amount of unmethylated templatepopulation compared to methylated template population and a second axisindicating a detected methylation signal. A detected methylation signalcan be any signal that corresponds to presence of a methylated targetnucleic acid including, for example, signals obtained in methodsdescribed herein. A calibration curve of the invention can be presentedin a variety of known formats including, without limitation, a hardcopy,graphical user interface of a computer, computer readable memory such asa magnetic disk, compact disc, random access memory or the like.

A calibration curve of the invention can be used to determine the extentof methylation for a sample under investigation. For example, amethylation signal can be measured for a sample using methods describedherein. The intensity of the signal can be compared to a calibrationcurve of the invention such that the extent of methylation correspondingto the signal intensity is determined.

As used herein, the term “reference genomic DNA” refers to a DNA derivedfrom the same individual organism as a sample to which it is compared.

The term “virtually unmethylated” used in reference to a first referencegenomic DNA refers to the absence of methyl containing bases or presenceof an amount of methyl containing bases that is negligible in routinedetection methods. The term can refer to a dilution of methylatedsequences by multiple rounds of amplification to create a sufficientlysmall ratio of methylated to unmethylated sequences that the methylatedsequences are rendered undetectable by routine detection methods. Asample having, for example, less than 1%, 0.05%, or 0.01% methylcontaining bases can be considered to be virtually unmethylated.

In particular embodiments a virtually unmethylated template populationcan be used to determine the limit of detection for a sample. In thisregard, signal detected from a virtually unmethylated templatepopulation provides a negative control, such that signals derivedtherefrom can be considered to constitute the lower limit of detectionand signals having higher intensity than those measured for thevirtually unmethylated template population are detectable andsignificant.

In some embodiments, as outlined below, the target sequences (or targetprobes, in some instances) may be attached to a solid support prior tocontact with the target probes (or to remove unhybridized target probes,etc.). In this embodiment, the target sequence may comprise apurification tag. By “purification tag” herein is meant a moiety whichcan be used to purify a strand of nucleic acid, usually via attachmentto a solid support as outlined herein. Suitable purification tagsinclude members of binding partner pairs. For example, the tag may be ahapten or antigen, which will bind its binding partner. In a preferredembodiment, the binding partner can be attached to a solid support asdepicted herein and in the figures. For example, suitable bindingpartner pairs include, but are not limited to: antigens (such asproteins (including peptides)) and antibodies (including fragmentsthereof (FAbs, etc.)); proteins and small molecules, includingbiotin/streptavidin; enzymes and substrates or inhibitors; otherprotein-protein interacting pairs; receptor-ligands; and carbohydratesand their binding partners. Nucleic acid—nucleic acid binding proteinspairs are also useful. In general, the smaller of the pair is attachedto the NTP for incorporation into the primer. Preferred binding partnerpairs include, but are not limited to, biotin (or imino-biotin) andstreptavidin, digoxyginin and Abs, and Prolinx™ reagents (seewww.prolinxinc.com/ie4/home.hmtl).

In a preferred embodiment, the binding partner pair comprises biotin orimino-biotin and streptavidin. Imino-biotin is particularly preferred asimino-biotin disassociates from streptavidin in pH 4.0 buffer whilebiotin requires harsh denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or90% formamide at 95° C.).

The present invention provides methods and compositions directed to themultiplex amplification and detection of methylated target sequencesutilizing target probes.

Accordingly, the invention provides a number of different primers andprobes. The probes and primers are nucleic acid as defined above.

Many of the probes and primers of the present invention are designed tohave at least a portion that binds substantially specifically to atarget nucleic acid (sometimes referred to herein as a bioactive agent(particularly in the case wherein the target analyte is not a nucleicacid) or a target specific portion). That is the probes are constructedso as to contain a target specific portion: a portion that binds to thetarget nucleic acid specifically, i.e. with high affinity. This targetspecific portion can be any type of molecule so long as it specificallybinds the target and can be attached to the rest of a target probe,namely a nucleic acid sequence that preferably includes an adaptersequence and at least one priming sequence.

In a preferred embodiment, the binding of the bioactive agent and thetarget nucleic acid is specific; that is, the bioactive agentspecifically binds to the target nucleic acid. By “specifically bind”herein is meant that the agent binds the target nucleic acid, withspecificity sufficient to differentiate between the target and othercomponents or contaminants of the test sample.

When nucleic acids are the target, the probes are designed to becomplementary to all or a portion (domain) of a target sequence (eitherthe target sequence of the sample or to other probe sequences, such asportions of amplicons, as is described below), such that hybridizationof the target sequence and the probes of the present invention occurs.As outlined below, this complementarity need not be perfect; there maybe any number of base pair mismatches which will interfere withhybridization between the target sequence and the single strandednucleic acids of the present invention. However, if the number ofmutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the bioactive agent portion of the probes aresufficiently complementary to all or part of the target sequences tohybridize under normal reaction conditions, and preferably give therequired specificity. In a preferred embodiment the probes have aportion that is exactly complementary to the target nucleic acids.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal, hereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (Tm) for the specific sequence at a defined ionic strengthand pH. The Tm is the temperature (under defined ionic strength, pH andnucleic acid concentration) at which 50% of the probes complementary tothe target hybridize to the target sequence at equilibrium (as thetarget sequences are present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 M sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of helix destabilizing agents such asformamide. The hybridization conditions may also vary when a non-ionicbackbone, i.e. PNA is used, as is known in the art. In addition,cross-linking agents may be added after target binding to cross-link,i.e. covalently attach, the two strands of the hybridization complex.

In a preferred embodiment, the target probes further comprise one ormore “adapter sequences” (sometimes referred to in the art as “zipcodes”) to allow the use of “universal arrays”. That is, arrays aregenerated that contain capture probes that are not target specific, butrather specific to individual artificial adapter sequences. The adaptersequences are added to the target probes, nested between the primingsequences (when two priming sequences are used) or “downstream” of asingle universal priming sequence, and thus are included in theamplicons. What is important is that the orientation of the primingsequence and the adapter sequence allows the amplification of theadapter sequence.

An “adapter sequence” is a sequence, generally exogeneous to the targetsequences, e.g. artificial, that is designed to be substantiallycomplementary (and preferably perfectly complementary) to a captureprobe of a detection array. Generally the capture probe is immobilizedto a solid support that can include microspheres or planar substratessuch as plastic or glass slides as described herein for array supports.In one embodiment the use of adapter sequences allow the creation ofmore “universal” surfaces; that is, one standard array, comprising afinite set of capture probes can be made and used in any application.The end-user can customize the array by designing different solubletarget probes, which, as will be appreciated by those in the art, isgenerally simpler and less costly. In a preferred embodiment, an arrayof different and usually artificial capture probes are made; that is,the capture probes do not have complementarity to known targetsequences. The adapter sequences can then be incorporated in the targetprobes.

As will be appreciated by those in the art, the length of the adaptersequences will vary, depending on the desired “strength” of binding andthe number of different adapters desired. In a preferred embodiment,adapter sequences range from about 6 to about 500 basepairs in length,with from about 8 to about 100 being preferred, and from about 10 toabout 25 being particularly preferred.

In a preferred embodiment, the adapter sequence uniquely identifies thetarget analyte to which the target probe binds. That is, while theadapter sequence need not bind itself to the target analyte, the systemallows for identification of the target analyte by detecting thepresence of the adapter. Accordingly, following a binding orhybridization assay and washing, the probes including the adapters areamplified. Detection of the adapter then serves as an indication of thepresence of the target analyte.

In one embodiment the adapter includes both an identifier region and aregion that is complementary to capture probes on a universal array asdescribed above. In this embodiment, the amplicon hybridizes to captureprobes on a universal array. Detection of the adapter is accomplishedfollowing hybridization with a probe that is complementary to theadapter sequence. Preferably the probe is labeled as described herein.

In general, unique adapter sequences are used for each unique targetanalyte. That is, the elucidation or detection of a particular adaptersequence allows the identification of the target analyte to which thetarget probe containing that adapter sequence bound. However, in somecases, it is possible to “reuse” adapter sequences and have more thanone target analyte share an adapter sequence.

In a preferred embodiment the adapters contain different sequences orproperties that are indicative of a particular target molecule. That is,each adapter uniquely identifies a target analyte. As described above,the adapters are amplified to form amplicons. The adapter is detected asan indication of the presence of the target analyte.

The use of adapters in combination with amplification following aspecific binding event allows for highly multiplexed reactions to beperformed.

Also, the probes are constructed so as to contain the necessary primingsite or sites for the subsequent amplification scheme. In a preferredembodiment the priming sites are universal priming sites. By “universalpriming site” or “universal priming sequences” herein is meant asequence of the probe that will bind a primer for amplification.

In a preferred embodiment, one universal priming sequence or site isused. In this embodiment, a preferred universal priming sequence is theRNA polymerase T7 sequence, that allows the T7 RNA polymerase make RNAcopies of the adapter sequence as outlined below.

In a preferred embodiment, for example when amplification methodsrequiring two primers such as PCR are used, each probe preferablycomprises an upstream universal priming site (UUP) and a downstreamuniversal priming site (DUP). Again, “upstream” and “downstream” are notmeant to convey a particular 5′-3′ orientation, and will depend on theorientation of the system. Preferably, only a single UUP sequence and asingle DUP sequence is used in a probe set, although as will beappreciated by those in the art, different assays or differentmultiplexing analysis may utilize a plurality of universal primingsequences. In addition, the universal priming sites are preferablylocated at the 5′ and 3′ termini of the target probe (or the ligatedprobe), as only sequences flanked by priming sequences will beamplified.

In addition, universal priming sequences are generally chosen to be asunique as possible given the particular assays and host genomes toensure specificity of the assay. However, as will be appreciated bythose in the art, sets of priming sequences/primers may be used; thatis, one reaction may utilize 500 target probes with a first primingsequence or set of sequences, and an additional 500 probes with a secondsequence or set of sequences.

As will be appreciated by those in the art, when two priming sequencesare used, the orientation of the two priming sites is different. Thatis, one PCR primer will directly hybridize to the first priming site,while the other PCR primer will hybridize to the complement of thesecond priming site. Stated differently, the first priming site is insense orientation, and the second priming site is in antisenseorientation.

The size of the primer and probe nucleic acid may vary, as will beappreciated by those in the art with each portion of the probe and thetotal length of the probe in general varying from 5 to 500 nucleotidesin length. Each portion is preferably between 10 and 100 beingpreferred, between 15 and 50 being particularly preferred, and from 10to 35 being especially preferred, depending on the use and amplificationtechnique. Thus, for example, the universal priming site(s) of theprobes are each preferably about 15-20 nucleotides in length, with 18being especially preferred. The adapter sequences of the probes arepreferably from 15-25 nucleotides in length, with 20 being especiallypreferred. The target specific portion of the probe is preferably from15-50 nucleotides in length. In addition, the primer may include anadditional amplification priming site. In a preferred embodiment theadditional amplification priming site is a T7 RNA polymerase primingsite.

Accordingly, the present invention provides first target probe sets. By“probe set” herein is meant a plurality of target probes that are usedin a particular multiplexed assay. In this context, plurality means atleast two, with more than 10 being preferred, depending on the assay,sample and purpose of the test. In one embodiment the probe set includesmore than 100, with more than 500 probes being preferred and more than1000 being particularly preferred. In a particularly preferredembodiment each probe contains at least 5000, with more than 10,000probes being most preferred.

Accordingly, the present invention provides first target probe sets thatcomprise at least a first universal priming site.

In a preferred embodiment, the target probe may also comprise a labelsequence, i.e. a sequence that can be used to bind label probes and issubstantially complementary to a label probe. This is sometimes referredto in the art as “sandwich-type” assays. That is, by incorporating alabel sequence into the target probe, which is then amplified andpresent in the amplicons, a label probe comprising primary (orsecondary) labels can be added to the mixture, either before addition tothe array or after. This allows the use of high concentrations of labelprobes for efficient hybridization. In one embodiment, it is possible touse the same label sequence and label probe for all target probes on anarray; alternatively, different target probes can have a different labelsequence. Similarly, the use of different label sequences can facilitatequality control; for example, one label sequence (and one color) can beused for one strand of the target, and a different label sequence (witha different color) for the other; only if both colors are present at thesame basic level is a positive called.

Thus, the present invention provides target probes that compriseuniversal priming sequences, bioactive agents (e.g. target specificportion(s)), adapter sequence(s), optionally an additional amplificationpriming sequence such as T7 RNA priming sequence and optionally labelsequences. These target probes are then added to the target sequences toform hybridization complexes. As will be appreciated by those in theart, when nucleic acids are the target, the hybridization complexescontain portions that are double stranded (the target-specific sequencesof the target probes hybridized to a portion of the target sequence) andportions that are single stranded (the ends of the target probescomprising the universal priming sequences and the adapter sequences,and any unhybridized portion of the target sequence, such as poly(A)tails, as outlined herein).

As will be appreciated by those in the art, the systems of the inventioncan take on a wide variety of configurations, including systems thatrely on the initial immobilization of the target analyte (solid phaseassays) and solution based assays.

Solid Phase Assays

In a preferred embodiment, the target analyte is immobilized on thesurface. That is, the target nucleic acids or target sequences areimmobilized on a substrate or capture surface. Attachment may beperformed in a variety of ways, as will be appreciated by those in theart, including, but not limited to, chemical or affinity capture (forexample, including the incorporation attachment moieties such asderivatized nucleotides such as AminoLink™ or biotinylated nucleotidesthat can then be used to attach the nucleic acid to a surface, as wellas affinity capture by hybridization), cross-linking, and electrostaticattachment, etc. When the target analyte is polyadenylated mRNA,supports comprising poly(T) sequences can be used. That is, anattachment moiety is attached to the target analyte that allows forattachment to the substrate. By “attachment moiety” is meant a moleculeor substance that mediates attachment of the target analyte to thesubstrate. In a preferred embodiment, affinity capture is used to attachthe nucleic acids to the support. For example, nucleic acids can bederivatized, for example with one member of a binding pair, and thesupport derivatized with the other member, i.e. a complementary member,of a binding pair. For example, the nucleic acids may be biotinylated(for example using enzymatic incorporation of biotinylated nucleotides,or by photoactivated cross-linking of biotin). In a preferred embodimentthe target nucleic acids are photobiotinylated In one preferredembodiment the target nucleic acids are photobiotinylated withPHOTOPROBE™ Biotin Reagents (Vector Laboratories). Biotinylated nucleicacids can then be captured on streptavidin-coated surfaces, as is knownin the art. In one embodiment the surfaces or supports are beads towhich the nucleic acids are attached, although other solid supports asdefined herein may also be used, e.g. microtiter plates. In aparticularly preferred embodiment the beads are magnetic beads.Similarly, other hapten-receptor combinations can be used, such asdigoxigenin and anti-digoxigenin antibodies. Alternatively, chemicalgroups can be added in the form of derivatized nucleotides, that canthen be used to add the nucleic acid to the surface.

Similarly, affinity capture utilizing hybridization can be used toattach nucleic acids to surface or bead. For example, a poly-A tract canbe attached by polymerization with terminal transferase, or via ligationof an oligo-A linker, as is known in the art. This then allows forhybridization with an immobilized poly-T tract. Alternatively, chemicalcrosslinking may be done, for example by photoactivated crosslinking ofthymidine to reactive groups, as is known in the art.

Preferred attachments are covalent, although even relatively weakinteractions (i.e. non-covalent) can be sufficient to attach a nucleicacid to a surface, if there are multiple sites of attachment per eachnucleic acid. Thus, for example, electrostatic interactions can be usedfor attachment, for example by having beads carrying the opposite chargeto the bioactive agent.

A preferred embodiment utilizes covalent attachment of the targetsequences to a support. As is known in the art, there are a wide varietyof methods used to covalently attach nucleic acids to surfaces. Apreferred embodiment utilizes the incorporation of a chemical functionalgroup into the nucleic acid, followed by reaction with a derivatized oractivated surface. Examples include, but are not limited to AminoLink™.

By “capture surface”, “target substrate” or “target support” or othergrammatical equivalents herein is meant any material to which a targetanalyte can be attached. The targets can be attached either directly orindirectly as described herein. As will be appreciated by those in theart, the number of possible substrates is very large. Possiblesubstrates include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, and a variety of other polymers. Preferably the substratesinclude microfuge tubes, i.e. Eppendorf tubes. In one embodiment thesubstrates include beads or microspheres. In one embodiment the beads ormicrospheres are magnetic, particularly for the capture of gDNA. In oneembodiment the substrates are derivatized to accommodate attachment ofthe target nucleic acids to the substrate.

The configuration of the target support is not crucial. What isimportant is that the target analytes are immobilized to the targetsupport and can be manipulated. That is, the support should be amenableto a variety of reactions as described herein. While the targetsubstrate can be flat (planar), other configurations of substrates maybe used as well; for example, target analytes can be attached to beadsor microspheres that can be deposited in reaction tubes or vessels orwells. That is, the target substrate may be microspheres to which thetarget analytes are attached. The microspheres can then be distributedon a surface. In some embodiments the surface contains reaction wellsinto which the beads are distributed, for example microtiter plates asare known in the art and as described herein.

Once the target analytes, i.e. genomic DNA or proteins, are applied toor immobilized on the surface, the target analytes are contacted withprobes for analyses, including detection or genotyping. That is, theappropriate probes necessary for detection of the target analyte or forthe methylation detection reactions are next introduced to theimmobilized sample.

For the assays described herein, the assays may be run under a varietyof experimental conditions, as will be appreciated by those in the art.A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding. Various blocking and washing stepsmay be utilized as is known in the art.

Following binding or hybridization of the bioactive agent portion of thetarget probe to the target analyte, unhybridized probes are removed by awashing step. In a preferred embodiment the wash step is a stringentwash step. That is, in the preferred embodiment of an enzymatic basedmutation detection reaction, once the probes have been introduced underconditions to favor hybridization with the appropriate nucleic acidsequences, a stringent wash step is conducted. This wash removesunhybridized probes and reduces the overall complexity of the mixture.It is this step that ensures the success of the overall multiplexedreaction.

As will be appreciated by those in the art, there are a wide variety ofdetection reactions that can be performed at this stage depending on thegoal of the assay. In a preferred embodiment, different target probesare made that span the region of the potentially methylated nucleotide.That is, target probes are designed to hybridize with a region spanningthe methylation position. If the target is cleaved, the methylationposition will be absent and the probe will not hybridize as efficientlyas it will with the uncleaved target. The wash step is done underconditions to wash away probes that hybridize to the cleaved targetThus, hybridization is indicative of uncleaved targets. Target probescan be applied to the target as one probe spanning the region of themethylated nucleotide or they may be the product of a modification ofthe probe, e.g. as a result of ligation or polymerase extension asdescribed herein.

In a preferred embodiment, when bisulfite is used as described above todetect methylation of the target nucleic acid, probes are designed tohybridize with either the “C” at the detection position or “U” at thedetection position. The presence of C indicates that the target wasmethylated and thus not converted to U upon incubation with bisulfite.

In a preferred embodiment when nucleic acids are the target, a pluralityof target probes (sometimes referred to herein as “readout targetprobes”) are used to identify the base at the detection position. Inthis embodiment, each different readout probe comprises a different baseat the position that will hybridize to the detection position of thetarget sequence (herein referred to as the readout or interrogationposition) and a different adapter sequence for each different readoutposition. In this way, differential hybridization of the readout targetprobes, depending on the sequence of the target, results inidentification of the base at the detection position. In thisembodiment, the readout probes are contacted with the array again underconditions that allow discrimination between match and mismatch, and theunhybridized probes are removed, etc.

Accordingly, by using different readout target probes, each with adifferent base at the readout position and each with a differentadapter, the identification of the base at the detection position iselucidated. Thus, in a preferred embodiment, a set of readout probes areused, each comprising a different base at the readout position.

In a preferred embodiment, each readout target probe has a differentadapter sequence. That is, readout target probes comprising adenine atthe readout position will have a first adapter, probes with guanine atthe readout position will have a second adapter, etc., such that eachtarget probe that hybridizes to the target sequence will bind to adifferent address on the array. This can allow the use of the same labelfor each reaction.

The number of readout target probes used will vary depending on the enduse of the assay.

In this embodiment, sensitivity to variations in stringency parametersare used to determine either the identity of the nucleotide(s) at thedetection position or the presence of a mismatch. As a preliminarymatter, the use of different stringency conditions such as variations intemperature and buffer composition to determine the presence or absenceof mismatches in double stranded hybrids comprising a single strandedtarget sequence and a probe is well known.

With particular regard to temperature, as is known in the art,differences in the number of hydrogen bonds as a function of basepairingbetween perfect matches and mismatches can be exploited as a result oftheir different Tms (the temperature at which 50% of the hybrid isdenatured). Accordingly, a hybrid comprising perfect complementaritywill melt at a higher temperature than one comprising at least onemismatch, all other parameters being equal. (It should be noted that forthe purposes of the discussion herein, all other parameters (i.e. lengthof the hybrid, nature of the backbone (i.e. naturally occurring ornucleic acid analog), the assay solution composition and the compositionof the bases, including G-C content are kept constant). However, as willbe appreciated by those in the art, these factors may be varied as well,and then taken into account.)

In general, as outlined herein, high stringency conditions are thosethat result in perfect matches remaining in hybridization complexes,while imperfect matches melt off. Similarly, low stringency conditionsare those that allow the formation of hybridization complexes with bothperfect and imperfect matches. High stringency conditions are known inthe art as outlined above.

As will be appreciated by those in the art, mismatch detection usingtemperature may proceed in a variety of ways.

Similarly, variations in buffer composition may be used to elucidate thepresence or absence of a mismatch at the detection position. Suitableconditions include, but are not limited to, formamide concentration.Thus, for example, “low” or “permissive” stringency conditions includeformamide concentrations of 0 to 10%, while “high” or “stringent”conditions utilize formamide concentrations of ≧40%. Low stringencyconditions include NaCl concentrations of ≧1 M, and high stringencyconditions include concentrations of ≦0.3 M. Furthermore, low stringencyconditions include MgCl₂ concentrations of ≧10 mM, moderate stringencyas 1-10 mM, and high stringency conditions include concentrations of ≦1mM.

In this embodiment, as for temperature, a plurality of readout probesmay be used, with different bases in the readout position and differentadapters. Running the assays under the permissive conditions andrepeating under stringent conditions will allow the elucidation of thebase at the detection position.

Thus, the washing is performed under stringency conditions which allowsformation of the first hybridization complex only between probes andcomplementary target sequences. As outlined above, stringency can becontrolled by altering a step parameter that is a thermodynamicvariable, including, but not limited to, temperature, formamideconcentration, salt concentration, chaotropic salt concentration, pH,organic solvent concentration, etc.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

In a preferred embodiment, the target sequence may be immobilized afterthe formation of the hybridization complexes, ligation complexes and/orligated complexes. That is, the probes can be added to the targets insolution, enzymes added as needed, etc. After the hybridizationcomplexes are formed and/or ligated, the hybridization complexes can beadded to supports comprising the binding partners and the unhybridizedprobes removed.

In this embodiment, particularly preferred binding ligand/bindingpartner pairs are biotin and streptavidin or avidin, antigens andantibodies.

As described above, once the hybridization complexes are formed,unhybridized probes are removed. This is important to increase the levelof multiplexing in the assay. In addition, as all target probes may formsome unpredictable structures that will complicate the amplificationusing the universal priming sequences. Thus to ensure specificity (e.g.that target probes directed to target sequences that are not present inthe sample are not amplified and detected), it is important to removeall the nonhybridized probes. As will be appreciated by those in theart, this may be done in a variety of ways, including methods based onthe target sequence, methods utilizing double stranded specificmoieties, and methods based on probe design and content. Preferably themethod includes a stringent wash step.

Once the non-hybridized probes (and additionally, if preferred, othersequences from the sample that are not of interest) are removed, thehybridization complexes are denatured and the target probes areamplified to form amplicons, which are then detected. This can be donein one of several ways as outlined below. In addition, as outlinedbelow, labels can be incorporated into the amplicons in a variety ofways.

Accordingly, this embodiment can be run in several modes. In a preferredembodiment, only a single probe is used, comprising (as outlinedherein), at least a first UUP, an adapter sequence, and atarget-specific portion, i.e. a target specific moiety or bioactiveagent. When nucleotides are the target molecule the target-specificportion includes nucleic acids comprising a first base at the readoutposition, and in some embodiments a DUP. This probe is contacted withthe target analyte under conditions (whether thermal or otherwise) suchthat specific binding occurs. In a preferred embodiment, when nucleicacids are the target, a hybridization complex is formed only when aperfect match between the detection position of the target and thereadout position of the probe is present. The non-hybridized ornon-bound probes are then removed as outlined herein. That is, after thewash step, only the properly hybridized probes should remain. In oneembodiment when nucleic acids are the target, the hybridized probes mustthen be separated from the captured sample nucleic acid. This is donevia a stringent wash step or denaturation step. The sample nucleic acidis left behind on the capture surface, and can be used again. In analternative embodiment, although not preferred, the hybridized probe isnot removed. it is not necessary to remove the probes when the primingsites and adapter sequences do not hybridize with the target. The probeis then amplified as outlined herein, and detected. In a preferredembodiment the amplified product(s), i.e. amplicons, are detected as anindication of the presence of the target analyte.

As noted above, the target sequence may be immobilized either before orafter the formation of the hybridization complex, but preferably it isimmobilized on a surface or support comprising the binding partner ofthe binding ligand prior to the formation of the hybridization complexwith the probe(s) of the invention. For example, a preferred embodimentutilizes binding partner coated reaction vessels such as eppendorf tubesor microtiter wells. Alternatively, the support may be in the form ofbeads, including magnetic beads. In this embodiment, the primary targetsequences are immobilized, the target probes are added to formhybridization complexes. Unhybridized probes are then removed throughwashing steps, and the bound probes (e.g. either target probes, ligatedprobes, or ligated RCA probes) are then eluted off the support, usuallythrough the use of elevated temperature or buffer conditions (pH, salt,etc.).

Once the non-hybridized probes (and additionally, if preferred, othersequences from the sample that are not of interest) are removed, thehybridization complexes are denatured and the target probes areamplified to form amplicons, which are then detected. This can be donein one of several ways, including PCR amplification and rolling circleamplification. Also, the probes can be amplified by known methods(exponential or linear amplification techniques such as PCR, Invader,ESPIA (also known as SPIA), T7), using the one or more priming sitesprovided on the probes. As noted herein, the probes are constructed soas to contain the necessary primer sites to permit this amplification.In a preferred embodiment, universal primers are used. Amplificationprovides the signal strength and dynamic range necessary for detectionof the mutation-detection probes. In addition, as outlined below, labelscan be incorporated into the amplicons in a variety of ways.

In a preferred embodiment, no ligation assay for genotyping is done,that is, no ligase is added. However, as will be appreciated by those inthe art, ligation reactions for other purposes may be done.

In a preferred embodiment, a linear amplification scheme known as ESPIA,or SPIA is applied. This amplification technique is disclosed in WO01/20035 A2 and U.S. Pat. No. 6,251,639, which are incorporated byreference herein. Generally, the method includes hybridizing chimericRNA/DNA amplification primers to the probes. Preferably the DNA portionof the probe is 3′ to the RNA. Optionally the method includeshybridizing a polynucleotide comprising a termination polynucleotidesequence to a region of the template that is 5′ with respect tohybridization of the composite primer to the template. Followinghybridization of the primer to the template, the primer is extended withDNA polymerase. Subsequently, the RNA is cleaved from the compositeprimer with an enzyme that cleaves RNA from an RNA/DNA hybrid.Subsequently, an additional RNA/DNA chimeric primer is hybridized to thetemplate such that the first extended primer is displaced from thetarget probe. The extension reaction is repeated, whereby multiplecopies of the probe sequence are generated.

In a preferred embodiment, the target amplification technique is PCR.The polymerase chain reaction (PCR) is widely used and described, andinvolves the use of primer extension combined with thermal cycling toamplify a target sequence; see U.S. Pat. Nos. 4,683,195 and 4,683,202,and PCR Essential Data, J. W. Wiley & sons, Ed. C. R. Newton, 1995, allof which are incorporated by reference.

In general, PCR may be briefly described as follows. The double strandedhybridization complex is denatured, generally by raising thetemperature, and then cooled in the presence of an excess of a PCRprimer, which then hybridizes to the first universal priming site. A DNApolymerase then acts to extend the primer with dNTPs, resulting in thesynthesis of a new strand forming a hybridization complex. The sample isthen heated again, to disassociate the hybridization complex, and theprocess is repeated. By using a second PCR primer for the complementarytarget strand that hybridizes to the second universal priming site,rapid and exponential amplification occurs. Thus PCR steps aredenaturation, annealing and extension. The particulars of PCR are wellknown, and include the use of a thermostable polymerase such as Taq Ipolymerase and thermal cycling. Suitable DNA polymerases include, butare not limited to, the Klenow fragment of DNA polymerase I, SEQUENASE1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29DNA polymerase.

The reaction is initiated by introducing the target probe comprising thetarget sequence to a solution comprising the universal primers, apolymerase and a set of nucleotides. By “nucleotide” in this contextherein is meant a deoxynucleoside-triphosphate (also calleddeoxynucleotides or dNTPs, e.g. dATP, dTTP, dCTP and dGTP). In someembodiments, as outlined below, one or more of the nucleotides maycomprise a detectable label, which may be either a primary or asecondary label. In addition, the nucleotides may be nucleotide analogs,depending on the configuration of the system. Similarly, the primers maycomprise a primary or secondary label.

Accordingly, the PCR reaction requires at least one PCR primer, apolymerase, and a set of dNTPs. As outlined herein, the primers maycomprise the label, or one or more of the dNTPs may comprise a label.

In a preferred embodiment, instead of using two primers (e.g. unlabeledT3 and biotin-labeled T7), a third primer (overlapping with T7, but isshorter than T7; labeled with another dye, for example, Fam) is added tothe PCR reaction. The PCR is first carried out at a lower stringentcondition for a certain cycles, i.e. 25-30 cycles, in which both thelonger and shorter PCR primers are annealed to the targets and generatePCR products; The PCR is then carried out at a higher stringentcondition for additional cycles, say additional 5-10 cycles. Under thishigher stringent condition, only the longer PCR primer can anneal to thetargets and further generate PCR products, while the shorter PCR primerwill not hybridize under this condition. Accordingly, for each of thetarget, two PCR products are generated with different PCR cycles andlabeled with different dyes. Since the two products are presented atdifferent concentrations in the final hybridization solution, the“shorter prime” signal can be used to measure the genes expressed athigh level without running into saturation problem, while the “longerprimer” signal is used to measure the genes expressed at low levelwithout losing the sensitivity. While the invention is described usingtwo primer variants, i.e. long and short prove, more than two variantscan be used. That is, preferably more than two primer variants are usedwith more than five being particularly preferred.

In addition, identical primers can be used, but the primers beardifferent labels. In this embodiment the ratio of the two labels in theproduct can be adjusted by varying the initial primer concentrations, sothere is no need to vary the PCR conditions.

In an alternative embodiment amplification can be performed using two ormore dye labeled dNTP (for the PCR) or NTP (for the IVT), pre-mixed atdifferent ratios. Accordingly, there is no need to vary the PCRconditions and PCR primer labeling. This method can also be used in theIVT step in gene expression monitoring using a direct hybridization withtotal RNA or mRNA, as a way to control the signal saturation problem. Assuch, detection of labels of different intensity serves to increase therange of detection of targets. That is, using less intense labels allowsfor detection of abundant targets without saturation while the use ofstronger labels serves to increase sensitivity allowing for detection ofless abundant targets.

In addition, the methods described above can be used in the final PCRstep in OLA-PCR genotyping as well, as long as the dyes are chosencorrectly such that they can be well-resolved by the hardware and/orsoftware of the systems. That is following the OLA reaction, theligation products can be amplified using primers as described above,i.e. either primer variants or differently labeled primers.

In a preferred embodiment, the methods of the invention include arolling circle amplification (RCA) step. This may be done in severalways. In one embodiment, either single target probes or ligated probescan be used in the genotyping part of the assay, followed by RCA insteadof PCR. Alternatively, and more preferably, the RCA reaction forms partof the genotyping reaction and can be used for both genotyping andamplification in the methods of the reaction.

In a preferred embodiment, the methods rely on rolling circleamplification. “Rolling circle amplification” is based on extension of acircular probe that has hybridized to a target sequence. A polymerase isadded that extends the probe sequence. As the circular probe has noterminus, the polymerase repeatedly extends the circular probe resultingin concatamers of the circular probe. As such, the probe is amplified.Rolling-circle amplification is generally described in Baner et al.(1998) Nuc. Acids Res. 26:5073-5078; Barany, F. (1991) Proc. Natl. Acad.Sci. USA 88:189-193; and Lizardi et al. (1998) Nat. Genet. 19:225-232,all of which are incorporated by reference in their entirety.

In general, RCA may be described in two ways, as generally depicted inFIGS. 9 and 10. First, as is outlined in more detail below, a singletarget probe is hybridized with a target nucleic acid. Each terminus ofthe probe hybridizes adjacently on the target nucleic acid and the OLAassay as described above occurs. When ligated, the probe is circularizedwhile hybridized to the target nucleic acid. Addition of a polyrneraseresults in extension of the circular probe. However, since the probe hasno terminus, the polymerase continues to extend the probe repeatedly.Thus results in amplification of the circular probe.

A second alternative approach involves a two step process. In thisembodiment, two ligation probes are initially ligated together, eachcontaining a universal priming sequence. A rolling circle primer is thenadded, which has portions that will hybridize to the universal primingsequences. The presence of the ligase then causes the original probe tocircularize, using the rolling circle primer as the polymerase primer,which is then amplified as above.

These embodiments also have the advantage that unligated probes need notnecessarily be removed, as in the absence of the target, no significantamplification will occur. These benefits may be maximized by the designof the probes; for example, in the first embodiment, when there is asingle target probe, placing the universal priming site close to the 5′end of the probe since this will only serve to generate short, truncatedpieces, without adapters, in the absence of the ligation reaction.

Accordingly, in an preferred embodiment, a single oligonucleotide isused both for OLA and as the circular template for RCA (referred toherein as a “padlock probe” or a “RCA probe”). That is, each terminus ofthe oligonucleotide contains sequence complementary to the targetnucleic acid and functions as an OLA primer as described above. That is,the first end of the RCA probe is substantially complementary to a firsttarget domain, and the second end of the RCA probe is substantiallycomplementary to a second target domain, adjacent to the first domain.Hybridization of the oligonucleotide to the target nucleic acid resultsin the formation of a hybridization complex. Ligation of the “primers”(which are the discrete ends of a single oligonucleotide) results in theformation of a modified hybridization complex containing a circularprobe i.e. an RCA template complex. That is, the oligonucleotide iscircularized while still hybridized with the target nucleic acid. Thisserves as a circular template for RCA. Addition of a primer and apolymerase to the RCA template complex results in the formation of anamplicon.

Labeling of the amplicon can be accomplished in a variety of ways; forexample, the polymerase may incorporate labeled nucleotides, oralternatively, a label probe is used that is substantially complementaryto a portion of the RCA probe and comprises at least one label is used,as is generally outlined herein.

The polymerase can be any polymerase, but is preferably one lacking 3′exonuclease activity (3′ exo⁻). Examples of suitable polymerase includebut are not limited to exonuclease minus DNA Polymerase I large (Klenow)Fragment, Phi29 DNA polymerase, Taq DNA Polymerase and the like. Inaddition, in some embodiments, a polymerase that will replicatesingle-stranded DNA (i.e. without a primer forming a double strandedsection) can be used. In addition, while some embodiments utilizeligase, such as in the OLA or RCA, in some embodiments amplificationalone is preferred. That is amplification is performed without a ligasestep and without including a ligase enzyme.

In a preferred embodiment, the RCA probe contains an adapter sequence asoutlined herein, with adapter capture probes on the array, for exampleon a microsphere when microsphere arrays are being used. Alternatively,unique portions of the RCA probes, for example all or part of thesequence corresponding to the target sequence, can be used to bind to acapture probe.

In a preferred embodiment, the padlock probe contains a restrictionsite. The restriction endonuclease site allows for cleavage of the longconcatamers that are typically the result of RCA into smaller individualunits that hybridize either more efficiently or faster to surface boundcapture probes. Thus, following RCA, the product nucleic acid iscontacted with the appropriate restriction endonuclease. This results incleavage of the product nucleic acid into smaller fragments. Thefragments are then hybridized with the capture probe that is immobilizedresulting in a concentration of product fragments onto the microsphere.Again, as outlined herein, these fragments can be detected in one of twoways: either labeled nucleotides are incorporated during the replicationstep, or an additional label probe is added.

Thus, in a preferred embodiment, the padlock probe comprises a labelsequence; i.e. a sequence that can be used to bind label probes and issubstantially complementary to a label probe. In one embodiment, it ispossible to use the same label sequence and label probe for all padlockprobes on an array; alternatively, each padlock probe can have adifferent label sequence.

The padlock probe also contains a priming site for priming the RCAreaction. That is, each padlock probe comprises a sequence to which aprimer nucleic acid hybridizes forming a template for the polymerase.The primer can be found in any portion of the circular probe. In apreferred embodiment, the primer is located at a discrete site in theprobe. In this embodiment, the primer site in each distinct padlockprobe is identical, e.g. is a universal priming site, although this isnot required. Advantages of using primer sites with identical sequencesinclude the ability to use only a single primer oligonucleotide to primethe RCA assay with a plurality of different hybridization complexes.That is, the padlock probe hybridizes uniquely to the target nucleicacid to which it is designed. A single primer hybridizes to all of theunique hybridization complexes forming a priming site for thepolymerase. RCA then proceeds from an identical locus within each uniquepadlock probe of the hybridization complexes.

In an alternative embodiment, the primer site can overlap, encompass, orreside within any of the above-described elements of the padlock probe.That is, the primer can be found, for example, overlapping or within therestriction site or the identifier sequence. In this embodiment, it isnecessary that the primer nucleic acid is designed to base pair with thechosen primer site.

Thus, the padlock probe of the invention contains at each terminus,sequences corresponding to OLA primers. The intervening sequence of thepadlock probe contain in no particular order, an adapter sequence and arestriction endonuclease site. In addition, the padlock probe contains aRCA priming site.

Thus, in a preferred embodiment the OLA/RCA is performed in solutionfollowed by restriction endonuclease cleavage of the RCA product. Thecleaved product is then applied to an array comprising beads, each beadcomprising a probe complementary to the adapter sequence located in thepadlock probe. The amplified adapter sequence correlates with aparticular target nucleic acid. Thus the incorporation of anendonuclease site allows the generation of short, easily hybridizablesequences. Furthermore, the unique adapter sequence in each rollingcircle padlock probe sequence allows diverse sets of nucleic acidsequences to be analyzed in parallel on an array, since each sequence isresolved on the basis of hybridization specificity.

Thus, the present invention provides for the generation of amplicons(sometimes referred to herein as secondary targets).

In a preferred embodiment, the amplicons are labeled with a detectionlabel. By “detection label” or “detectable label” herein is meant amoiety that allows detection. This may be a primary label or a secondarylabel. Accordingly, detection labels may be primary labels (i.e.directly detectable) or secondary labels (indirectly detectable).

In a preferred embodiment, the detection label is a primary label. Aprimary label is one that can be directly detected, such as afluorophore. In general, labels fall into three classes: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic,electrical, thermal labels; and c) colored or luminescent dyes. Labelscan also include enzymes (horseradish peroxidase, etc.) and magneticparticles. Preferred labels include chromophores or phosphors but arepreferably fluorescent dyes. Suitable dyes for use in the inventioninclude, but are not limited to, fluorescent lanthanide complexes,including those of Europium and Terbium, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,quantum dots (also referred to as “nanocrystals”: see U.S. Ser. No.09/315,584, hereby incorporated by reference), pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, Cy dyes (Cy3, Cy5,etc.), alexa dyes, phycoerythin, bodipy, and others described in the 6thEdition of the Molecular Probes Handbook by Richard P. Haugland, herebyexpressly incorporated by reference.

In a preferred embodiment, a secondary detectable label is used. Asecondary label is one that is indirectly detected; for example, asecondary label can bind or react with a primary label for detection,can act on an additional product to generate a primary label (e.g.enzymes), or may allow the separation of the compound comprising thesecondary label from unlabeled materials, etc. Secondary labels include,but are not limited to, one of a binding partner pair such asbiotin/streptavidin; chemically modifiable moieties; nucleaseinhibitors, enzymes such as horseradish peroxidase, alkalinephosphatases, lucifierases, etc.

In a preferred embodiment, the secondary label is a binding partnerpair. For example, the label may be a hapten or antigen, which will bindits binding partner. In a preferred embodiment, the binding partner canbe attached to a solid support to allow separation of extended andnon-extended primers. For example, suitable binding partner pairsinclude, but are not limited to: antigens (such as proteins (includingpeptides)) and antibodies (including fragments thereof (FAbs, etc.));proteins and small molecules, including biotin/streptavidin; enzymes andsubstrates or inhibitors; other protein-protein interacting pairs;receptor-ligands; and carbohydrates and their binding partners. Nucleicacid—nucleic acid binding proteins pairs are also useful. In general,the smaller of the pair is attached to the NTP for incorporation intothe primer. Preferred binding partner pairs include, but are not limitedto, biotin (or imino-biotin) and streptavidin, digeoxinin and Abs, andProlinx™ reagents (see www.prolinxinc.com/ie4/home.hmtl).

In a preferred embodiment, the binding partner pair comprises biotin orimino-biotin and streptavidin. Imino-biotin is particularly preferred asimino-biotin disassociates from streptavidin in pH 4.0 buffer whilebiotin requires harsh denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or90% formamide at 95° C.).

In a preferred embodiment, the binding partner pair comprises a primarydetection label (for example, attached to the NTP and therefore to theamplicon) and an antibody that will specifically bind to the primarydetection label. By “specifically bind” herein is meant that thepartners bind with specificity sufficient to differentiate between thepair and other components or contaminants of the system. The bindingshould be sufficient to remain bound under the conditions of the assay,including wash steps to remove non-specific binding. In someembodiments, the dissociation constants of the pair will be less thanabout 10⁻⁴-10⁻⁶ M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ beingpreferred and less than about 10⁻⁷-10⁻⁹ M⁻¹ being particularlypreferred.

In a preferred embodiment, the secondary label is a chemicallymodifiable moiety. In this embodiment, labels comprising reactivefunctional groups are incorporated into the nucleic acid. The functionalgroup can then be subsequently labeled with a primary label. Suitablefunctional groups include, but are not limited to, amino groups, carboxygroups, maleimide groups, oxo groups and thiol groups, with amino groupsand thiol groups being particularly preferred. For example, primarylabels containing amino groups can be attached to secondary labelscomprising amino groups, for example using linkers as are known in theart; for example, homo- or hetero-bifunctional linkers as are well known(see 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).

As outlined herein, labeling can occur in a variety of ways, as will beappreciated by those in the art. In general, labeling can occur in oneof three ways: labels are incorporated into primers such that theamplification reaction results in amplicons that comprise the labels;labels are attached to dNTs and incorporated by the polymerase into theamplicons; or the amplicons comprise a label sequence that is used tohybridize a label probe, and the label probe comprises the labels. Itshould be noted that in the latter case, the label probe can be addedeither before the amplicons are contacted with an array or afterwards.

A preferred embodiment utilizes one primer comprising a biotin, that isused to bind a fluorescently labeled streptavidin.

In a preferred embodiment following amplification, the amplicons aresubjected to an additional amplification step. Preferably the additionalamplification step is a T7 RNA polymerase reaction, although T7amplification also can be the primary amplification step. The advantageof following the amplification step with an additional amplificationstep such as the T7 RNA Polymerase reaction is that up to one hundredfold or more nucleic acid is generated therefore increasing the level ofmultiplexing.

As described above, the probes include T7 RNA polymerase priming sitesfor this additional step. In some embodiments this priming sitecomprises the universal priming site. Following amplification with T7RNA polymerase, the resulting RNA contains a zip code and a universalprimer that is allele specific. The resulting material is then detected.

In addition, in one embodiment of allele discrimination, the primers caninclude either T7 or T3 priming sites that are specific for a particularallele. That is, in addition to allele specific primers and universalpriming sites for universal amplification, the primers may also includeselective amplification priming site, such as either T7 or T3. By“selective” is meant a priming site that allows for allele selectiveamplification and discrimination. In the case of T7 or T3, the primingsites serve as promoters for RNA polymerases. For example, T3 promotersequence is selective for a first allele and T7 is selective for asecond allele. Thus, following the SNP specific OLA assay, includingprimary amplification, in vitro transcription (IVT) is performed in twoseparate reactions. Each reaction is carried out with one particular RNAPolymerase (T3 or T7). Preferably the reactions are carried out in thepresence of a label. The products of the reactions are then detected.

In a preferred embodiment, following treatment of target nucleic acidswith bisulfite, the modified target is contacted with target probesdesigned to be complementary to locus sequence and either the C or U atthe potentially methylated position. Preferably one of the primersincludes a first priming site and the other primer includes a secondpriming site. Such priming sites are exemplified, without limitation, byT3 and T7. Then the primers are replicated, either by amplification oran in vitro transcription reaction is preformed with T3 or T7 RNAPolymerase respectively, in the presence of different labels. Labeledamplification products are analyzed for the presence of either of thelabels or both of the labels. In this way, the invention provides for amethod of determining if zero, one or both chromosomes are methylated.That is, the results will demonstrate either the first label(corresponding to the first probe complementary to the C at thedetection position), the second label (corresponding to the second probecomplementary to the U at the detection position), or both.

In an additional embodiment the reactions are carried out with separatelabels. That is, one label that corresponds to each reaction, i.e. Cy3and Cy5 for T3 and T7, reactions, respectively, is included in thereactions. When two labels are used, the products of the reactions canbe pooled and then detected by any of the detection methods describedherein.

In one embodiment the amplicons are detected by hybridization to anarray. The array can be an ordered array or a random array as describedherein. In addition, the array can be a liquid array. That is, the arraycan be a solution-phase array and detection is accomplished in a FACS,for example. In a preferred embodiment the detection array is a randomBeadArray™.

In addition the following methods that are typically used forgenotyping, find use in methylation detection. That is, the presentinvention also provides methods for accomplishing genotyping of nucleicacids, including cDNA and genomic DNA. In general, this method can bedescribed as follows, as is generally described in WO 00/63437, herebyexpressly incorporated by reference. Genomic DNA is prepared from samplecells (and generally cut into smaller segments, for example throughshearing or enzymatic treatment with enzymes such as DNAse I, as is wellknown in the art). In some embodiments a restriction enzyme is used. Inthis embodiment the restriction cleavage site and the target selectionscan be designed based on genomic sequences, i.e. using computer aidedanalysis, such that the un-methylated genomic regions will be digestedby a methylation selective or discriminatory enzyme as described hereinand will not be immobilized to the solid support or only part of thetarget will be immobilized.

Using any number of techniques, as are outlined below, the genomicfragments are attached, either covalently or securely, to a support suchas beads or reaction wells (eppendorf tubes, microtiter wells, etc.).Any number of different reactions can then be done as outlined below todetect methylated target nucleic acids, and the reaction products fromthese reactions are released from the support, amplified as necessaryand added to an array of capture probes as outlined herein. In general,the methods described herein relate to the detection of methylatedtarget nucleotides Universal primers can also be included as necessary.

These techniques fall into five general categories: (1) techniques thatrely on traditional hybridization methods that utilize the variation ofstringency conditions (temperature, buffer conditions, etc.) todistinguish nucleotides at the detection position; (2) extensiontechniques that add a base (“the base”) to basepair with the nucleotideat the detection position; (3) ligation techniques, that rely on thespecificity of ligase enzymes (or, in some cases, on the specificity ofchemical techniques), such that ligation reactions occur preferentiallyif perfect complementarity exists at the detection position; (4)cleavage techniques, that also rely on enzymatic or chemical specificitysuch that cleavage occurs preferentially if perfect complementarityexists; and (5) techniques that combine these methods. See generally WO00/63437, incorporated by reference in its entirety.

As above, if required, the target genomic sequence is prepared usingknown techniques, and then attached to a solid support as definedherein. These techniques include, but are not limited to, enzymaticattachment, chemical attachment, photochemistry or thermal attachmentand absorption.

In a preferred embodiment, as outlined herein, enzymatic techniques areused to attach the genomic DNA to the support. For example, terminaltransferase end-labeling techniques can be used as outlined above; seeHermanson, Bioconjugate Techniques, San Diego, Academic Press, pp640-643). In this embodiment, a nucleotide labeled with a secondarylabel (e.g. a binding ligand) is added to a terminus of the genomic DNA;supports coated or containing the binding partner can thus be used toimmobilize the genomic DNA. Alternatively, the terminal transferase canbe used to add nucleotides with special chemical functionalities thatcan be specifically coupled to a support. Similarly, random-primedlabeling or nick-translation labeling (supra, pp. 640-643) can also beused.

In a preferred embodiment, chemical labeling (supra, pp. 6444-671) canbe used. In this embodiment, bisulfite-catalyzed transamination,sulfonation of cytosine residues, bromine activation of T, C and Gbases, periodate oxidation of RNA or carbodiimide activation of 5′phosphates can be done.

In a preferred embodiment, photochemistry or heat-activated labeling isdone (supra, p162-166). Thus for example, aryl azides and nitrenespreferably label adenosines, and to a less extent C and T (Aslam et al.,Bioconjugation: Protein Coupling Techniques for Biomedical Sciences; NewYork, Grove's Dictionaries, 833 pp.). Psoralen or angelicin compoundscan also be used (Aslam, p492, supra). The preferential modification ofguanine can be accomplished via intercalation of platinum complexes(Aslam, supra).

In a preferred embodiment, the genomic DNA can be absorbed on positivelycharged surfaces, such as an amine coated solid phase. The genomic DNAcan be cross-linked to the surface after physical absorption forincreased retention (e.g. PEI coating and glutaraldehyde cross-linking;Aslam, supra, p. 485).

In a preferred embodiment, direct chemical attached or photocrosslinkingcan be done to attach the genomic DNA to the solid phase, by usingdirect chemical groups on the solid phase substrate. For example,carbodiimide activation of 5′ phosphates, attachment to exocyclic amineson DNA bases, and psoralen can be attached to the solid phase forcrosslinking to the DNA.

Once added to the support, the target genomic sequence can be used in avariety of reactions for a variety of reasons. For example, in apreferred embodiment, genotyping reactions are done. Similarly, thesereactions can also be used to detect the presence or absence of a targetgenomic sequence. In addition, in any reaction, quantitation of theamount of a target genomic sequence may be done. While the discussionbelow focuses on genotyping reactions, the discussion applies equally todetecting the presence of target sequences and/or their quantification.

As will be appreciated by those in the art, the reactions describedbelow can take on a wide variety of formats. In one embodiment, genomicDNA is attached to a solid support, and probes comprising universalprimers are added to form hybridization complexes, in a variety offormats as outlined herein. The non-hybridized probes are then removed,and the hybridization complexes are denatured This releases the probes(which frequently have been altered in some way). They are thenamplified and added to an array of capture probes. In a preferredembodiment, non-hybridized primers are removed prior to the enzymaticstep. Several embodiments of this have been described above.Alternatively, genomic DNA is attached to a solid support, andmethylation reactions are done in formats that can allow amplificationas well, either during the reaction (e.g. through the use of heatcycling) or after, without the use of universal primers. Thus, forexample, when labeled probes are used, they can be hybridized to theimmobilized genomic DNA, unbound materials removed, and then eluted andcollected to be added to arrays. This may be repeated for amplificationpurposes, with the elution fractions pooled and added to the array. Inaddition, alternative amplification schemes such as extending a productof the invasive cleavage reaction (described below) to include universalprimers or universal primers and adapters can be performed. In oneembodiment this allows the reuse of immobilized target sequences with adifferent set or sets of target probes.

In some embodiments, amplification of the product of the genotypingreactions is not necessary. For example, in genomes of less complexity,e.g. bacterial, yeast and Drosophila, detectable signal is achievedwithout the need for amplification. This is particularly true whenprimer extension is performed and more than one base is added to theprobe, as is more fully outlined below.

In a preferred embodiment, straight hybridization methods are used toelucidate the identity of the base at the detection position. Generallyspeaking, these techniques break down into two basic types of reactions:those that rely on competitive hybridization techniques, and those thatdiscriminate using stringency parameters and combinations thereof.

In a preferred embodiment, the use of competitive hybridization probesis done to elucidate either the identity of the nucleotide(s) at thedetection position or the presence of a mismatch. For example,sequencing by hybridization has been described (Drmanac et al., Genomics4:114 (1989); Koster et al., Nature Biotechnology 14:1123 (1996); U.S.Pat. Nos. 5,525,464; 5,202,231 and 5,695,940, among others, all of whichare hereby expressly incorporated by reference in their entirety).

As outlined above, in a preferred embodiment, a plurality of readoutprobes are used to identify the base at the detection position. In thisembodiment, each different readout probe comprises either a differentdetection label (which, as outlined below, can be either a primary labelor a secondary label) or a different adapter, and a different base atthe position that will hybridize to the detection position of the targetsequence (herein referred to as the readout position) such thatdifferential hybridization will occur.

Accordingly, in some embodiments, a detectable label is incorporatedinto the readout probe. In a preferred embodiment, a set of readoutprobes are used, each comprising a different base at the readoutposition. In some embodiments, each readout probe comprises a differentlabel, that is distinguishable from the others. For example, a firstlabel may be used for probes comprising adenosine at the readoutposition, a second label may be used for probes comprising guanine atthe readout position, etc. In a preferred embodiment, the length andsequence of each readout probe is identical except for the readoutposition, although this need not be true in all embodiments.

In one embodiment, the probes used as readout probes are “MolecularBeacon” probes as are generally described in Whitcombe et al., NatureBiotechnology 17:804 (1999), hereby incorporated by reference. As isknown in the art, Molecular Beacon probes form “hairpin” typestructures, with a fluorescent label on one end and a quencher on theother. In the absence of the target sequence, the ends of the hairpinhybridize, causing quenching of the label. In the presence of a targetsequence, the hairpin structure is lost in favor of target sequencebinding, resulting in a loss of quenching and thus an increase insignal.

In a preferred embodiment, extension genotyping is done. In thisembodiment, any number of techniques are used to add a nucleotide to thereadout position of a probe hybridized to the target sequence adjacentto the detection position. By relying on enzymatic specificity,preferentially a perfectly complementary base is added. All of thesemethods rely on the enzymatic incorporation of nucleotides at thedetection position. This may be done using chain terminating dNTPs, suchthat only a single base is incorporated (e.g. single base extensionmethods), or under conditions that only a single type of nucleotide isadded followed by identification of the added nucleotide (extension andpyrosequencing techniques).

In a preferred embodiment, single base extension (SBE; sometimesreferred to as “misequencing”) is used to determine the identity of thebase at the detection position. SBE utilizes an extension primer with atleast one adapter sequence that hybridizes to the target nucleic acidimmediately adjacent to the detection position, to form a hybridizationcomplex. A polymerase (generally a DNA polymerase) is used to extend the3′ end of the primer with a nucleotide analog labeled with a detectionlabel as described herein. Based on the fidelity of the enzyme, anucleotide is only incorporated into the readout position of the growingnucleic acid strand if it is perfectly complementary to the base in thetarget strand at the detection position. The nucleotide may bederivatized such that no further extensions can occur, so only a singlenucleotide is added. Once the labeled nucleotide is added, detection ofthe label proceeds as outlined herein. Again, amplification in this caseis accomplished through cycling or repeated rounds of reaction/elution,although in some embodiments amplification is not necessary.

The reaction is initiated by introducing the hybridization complexcomprising the target genomic sequence on the support to a solutioncomprising a first nucleotide. In general, the nucleotides comprise adetectable label, which may be either a primary or a secondary label. Inaddition, the nucleotides may be nucleotide analogs, depending on theconfiguration of the system. For example, if the dNTPs are added insequential reactions, such that only a single type of dNTP can be added,the nucleotides need not be chain terminating. In addition, in thisembodiment, the dNTPs may all comprise the same type of label.

Alternatively, if the reaction comprises more than one dNTP, the dNTPsshould be chain terminating, that is, they have a blocking or protectinggroup at the 3′ position such that no further dNTPs may be added by theenzyme. As will be appreciated by those in the art, any number ofnucleotide analogs may be used, as long as a polymerase enzyme willstill incorporate the nucleotide at the readout position. Preferredembodiments utilize dideoxy-triphosphate nucleotides (ddNTPs) andhalogenated dNTPs. Generally, a set of nucleotides comprising ddATP,ddCTP, ddGTP and ddTTP is used, each with a different detectable label,although as outlined herein, this may not be required. Alternativepreferred embodiments use acyclo nucleotides (NEN). These chainterminating nucleotide analogs are particularly good substrates for Deepvent (exo⁻) and thermosequenase.

In addition, as will be appreciated by those in the art, the single baseextension reactions of the present invention allow the preciseincorporation of modified bases into a growing nucleic acid strand.Thus, any number of modified nucleotides may be incorporated for anynumber of reasons, including probing structure-function relationships(e.g. DNA:DNA or DNA:protein interactions), cleaving the nucleic acid,crosslinking the nucleic acid, incorporate mismatches, etc.

As will be appreciated by those in the art, the configuration of themethylation SBE system can take on several forms.

In addition, since unextended primers do not comprise labels, theunextended primers need not be removed. However, they may be, ifdesired, as outlined below; for example, if a large excess of primersare used, there may not be sufficient signal from the extended primerscompeting for binding to the surface.

Alternatively, one of skill in the art could use a single label andtemperature to determine the identity of the base; that is, the readoutposition of the extension primer hybridizes to a position on the captureprobe. However, since the three mismatches will have lower Tms than theperfect match, the use of temperature could elucidate the identity ofthe detection position base.

In a preferred embodiment, the determination of the identity of the baseat the detection position of the target sequence proceeds using invasivecleavage technology. As outlined above for amplification, invasivecleavage techniques rely on the use of structure-specific nucleases,where the structure can be formed as a result of the presence or absenceof a mismatch. Generally, invasive cleavage technology may be describedas follows. A target nucleic acid is recognized by two distinct probes.A first probe, generally referred to herein as an “invader” probe, issubstantially complementary to a first portion of the target nucleicacid. A second probe, generally referred to herein as a “signal probe”,is partially complementary to the target nucleic acid; the 3′ end of thesignal oligonucleotide is substantially complementary to the targetsequence while the 5′ end is non-complementary and preferably forms asingle-stranded “tail” or “arm”. The non-complementary end of the secondprobe preferably comprises a “generic” or “unique” sequence, frequentlyreferred to herein as a “detection sequence”, that is used to indicatethe presence or absence of the target nucleic acid, as described below.The detection sequence of the second probe may comprise at least onedetectable label (for cycling purposes), or preferably comprises one ormore universal priming sites and/or an adapter sequence. Alternativemethods have the detection sequence functioning as a target sequence fora capture probe, and thus rely on sandwich configurations using labelprobes.

Hybridization of the first and second oligonucleotides near or adjacentto one another on the target genomic nucleic acid forms a number ofstructures.

Accordingly, the present invention provides methods of determining theidentity of a base at the detection position of a target sequence. Inthis embodiment, the target sequence comprises, 5′ to 3′, a first targetdomain comprising an overlap domain comprising at least a nucleotide inthe detection position, and a second target domain contiguous with thedetection position. A first probe (the “invader probe”) is hybridized tothe first target domain of the target sequence. A second probe (the“signal probe”), comprising a first portion that hybridizes to thesecond target domain of the target sequence and a second portion thatdoes not hybridize to the target sequence, is hybridized to the secondtarget domain. If the second probe comprises a base that is perfectlycomplementary to the detection position a cleavage structure is formed.The addition of a cleavage enzyme, such as is described in U.S. Pat.Nos. 5,846,717; 5,614,402; 5,719,029; 5,541,311 and 5,843,669, all ofwhich are expressly incorporated by reference, results in the cleavageof the detection sequence from the signaling probe. This then can beused as a target sequence in an assay complex.

In addition, as for a variety of the techniques outlined herein,unreacted probes (i.e. signaling probes, in the case of invasivecleavage), may be removed using any number of techniques. For example,the use of a binding partner coupled to a solid support comprising theother member of the binding pair can be done. Similarly, after cleavageof the primary signal probe, the newly created cleavage products can beselectively labeled at the 3′ or 5′ ends using enzymatic or chemicalmethods.

Again, as outlined above, the detection of the invasive cleavagereaction can occur directly, in the case where the detection sequencecomprises at least one label, or indirectly, using sandwich assays,through the use of additional probes; that is, the detection sequencescan serve as target sequences, and detection may utilize amplificationprobes, capture probes, capture extender probes, label probes, and labelextender probes, etc. In one embodiment, a second invasive cleavagereaction is performed on solid-phase thereby making it easier performmultiple reactions.

In addition, as for most of the techniques outlined herein, thesetechniques may be done for the two strands of a double-stranded targetsequence. The target sequence is denatured, and two sets of probes areadded: one set as outlined above for one strand of the target, and aseparate set for the other strand of the target.

Thus, the invasive cleavage reaction requires, in no particular order,an invader probe, a signaling probe, and a cleavage enzyme.

It is also possible to combine two or more of these techniques to dogenotyping, quantification, detection of sequences, etc., again asoutlined in WO 00/63437, expressly incorporated by reference, includingcombinations of competitive hybridization and extension, particularlySBE; a combination of competitive hybridization and invasive cleavage;invasive cleavage and ligation; a combination of invasive cleavage andextension reactions; a combination of OLA and SBE; a combination of OLAand PCR; a combination of competitive hybridization and ligation; and acombination of competitive hybridization and invasive cleavage.

Solution Phase Assays

Alternatively, the assays of the invention can be run in solution,followed by detection of the amplicons, either by the addition of theamplicons to an array or utilizing other methods as outlined herein(mass spectroscopy, electrophoresis, etc.) as outlined herein. In thisembodiment, a variety of methods can be used to remove unhybridizedtarget probes, as outlined in WO 00/63437, expressly incorporated byreference herein.

For example, if the target analyte is not immobilized, separationmethods based on the differences between single-stranded anddouble-stranded nucleic acids may be done. For example, there are avariety of double-stranded specific moieties known, that preferentiallyinteract with double-stranded nucleic acids over single stranded nucleicacids. For example, there are a wide variety of intercalators known,that insert into the stacked basepairs of double stranded nucleic acid.Two of the best known examples are ethidium bromide and actinomycin D.Similarly, there are a number of major groove and minor groove bindingproteins which can be used to distinguish between single stranded anddouble stranded nucleic acids. Similar to the poly(T) embodiment, thesemoieties can be attached to a support such as magnetic beads and used topreferentially bind the hybridization complexes, to remove thenon-hybridized target probes and target sequences during washing steps.The hybridization complexes are then released from the beads using adenaturation step such as a thermal step.

In the case where the OLA reaction is done, an additional embodiment,depicted in FIG. 8, may be done to remove unhybridized primers. In thisembodiment, a nuclease inhibitor is added to the 3′ end of thedownstream ligation probe, which does not comprise the adapter sequence.Thus, any nucleic acids that do not contain the inhibitors (includingboth the 5′ unligated probe and the target sequences themselves) will bedigested upon addition of a 3′-exonuclease. The ligation products areprotected from exo I digestion by including, for example,4-phosphorothioate residues at their 3′ terminus, thereby, renderingthem resistant to exonuclease digestion. The unligated detectionoligonucleotides are not protected and are digested. Since the 5′upstream ligation probe carries the adapter sequence, the unligateddownstream probe, which does carry the nuclease inhibitor and is thusalso not digested, does not bind to the array and can be washed away.The nuclease inhibitors may also be used in non-OLA utilities as well.

Suitable nuclease inhibitors are known in the art and comprise thiolnucleotides. In this embodiment, suitable 3′-exonucleases include, butare not limited to, exo I, exo III, exo VII, and 3′-5′exophosphodiesterases.

Following the amplification procedure, there is present sufficientnucleic acid material to detect the results of the genotyping assaysthrough conventional means. In the preferred embodiment, the probes usedin the mutation detection reaction also contain address sequences.During the amplification process, the address sequences used to read outthe results are simultaneously amplified with the mutation-detectionprobes. When the amplified material is applied to a detection substrate,such as an array where complementary address sequences are provided, theamplified nucleic acid probes are then detected by known methods.

Combination Techniques

Other preferred configurations of the system are set forth in U.S. Ser.No. 10/177,727, filed Jun. 20, 2002 now published U.S. application2003/0200489 and Ser. No. 10/194,958, filed Jul. 12, 2002, now U.S. Pat.No. 7,582,240 both of which are expressly incorporated herein byreference.

The following methods are generally directed to methods of alleledetection and find use in detecting methylated target nucleic acids whenthe target nucleic acids are subjected to the methylation selectivemethods described herein. Accordingly, following a methylation selectivestep as described herein, and immobilization of the modified target, inone embodiment the target nucleic acids are contacted with allelespecific probes under stringent annealing conditions. Non-hybridizedprobes are removed by a stringent wash. Subsequently the hybridizedprobes or primers are contacted with an enzyme such as a polymerase inthe presence of labeled ddNTP forming a modified primer. Preferably thelabel is a purification tag as described herein. The ddNTP is onlyincorporated into the primer that is perfectly complementary to thetarget nucleic acid. The modified primer is then eluted from theimmobilized target nucleic acid, and contacted with amplificationprimers to form amplicons. In one embodiment the eluted primer ispurified by binding to a binding partner for the affinity tag. Then thepurified and modified primer is contacted with amplification primers foramplification, forming amplicons. The amplicons are then detected as anindication of the presence of the particular target nucleic acid, e.g.determining whether the target is methylated or not.

In a preferred embodiment, the allele specific primer also includes anadapter sequence and priming sequences as described herein.

Alternatively, allele detection proceeds as a result of allele specificamplification. That is, at least one of the priming sequences on theprimers for each allele is specific for a particular allele, ormethylation state of the target. Thus, following hybridization of theprimers and removal of the unhybridized primers, one of the alleles willbe identified. Following addition of the respective amplificationprimers, only one set of the primers will hybridize with the primingsequences. Thus, only one of the sets of primers will generate anamplicon. In a preferred embodiment, each of the sets of primers islabeled with distinct label. Because only one of the sets will beamplified, detection of a label provides an indication of the primerthat was amplified. This, in turn identifies the nucleotide at thedetection position.

In an alternative embodiment the target nucleic acid is first contactedwith a first target specific probe under stringent annealing conditionsand a first extension reaction is performed with either dNTPs or ddNTPSforming a first extension product. The first target specific probe inthis embodiment is either a locus specific probe or an allele specificprobe. This step reduces the complexity of the sample. Subsequently thefirst extension product is contacted with a second probe that has thesame sequence as a portion of the target sequence, i.e. the second probeis complementary to the extension product, and again can be either anallele specific probe or a locus specific probe. Following hybridizationof the second probe, a second extension reaction is performed.

In a preferred embodiment the primers for the first and second extensionreaction also include amplification priming sites. Preferably theamplification priming sites are universal priming sites as describedherein. Accordingly, the resulting extension product is amplified (theamplification component of the multiplexing scheme). The resultingdouble stranded product is then denatured and either of the strands isused as a template for a single base extension (SBE) reaction asdescribed in more detail below (the specificity component). In the SBEreaction, chain terminating nucleotides such as ddTNPs are used assubstrates for the polymerase and are incorporated into a target probethat is hybridized to the single stranded amplicon template adjacent tothe interrogation position. Preferably the ddNTPs are labeled asdescribed below. Preferably, the ddNTPs are discretely labeled such thatthey can be discriminated in the detection step.

In an alternative embodiment a first biotinylated or otherwise taggedprobe is hybridized with a target nucleic acid and a first extensionreaction is performed. The primer or probe is either an allele specificor locus specific probe. The extended product is then purified from themixture by the tag. Again, this serves as the complexity reduction step.Subsequently, a second primer is hybridized to the first extensionproduct and a second extension reaction is performed, preferably in anallele specific manner, i.e. with discriminatory probes that arespecific for, each allele. This represents the specificity step.Preferably, both of the primers used in the extension reactions containuniversal priming sites. Thus, universal primers can be added foruniversal amplification of the extension products (the amplificationcomponent. In a preferred embodiment, each allele specific primerincludes a distinct amplification priming site. Thus, following allelediscrimination, only one of the primers can be used for amplification,resulting in allele specific amplification. Preferably the amplificationprimers contain discrete labels, which again allows for detection ofwhich particular primers served as amplification templates. This, again,identifies the particular allele to be detected. In an additionalpreferred embodiment, at least one of the primers includes an adaptersequence as outlined below.

In an alternative embodiment tagged, i.e. biotinylated, primers arehybridized with a target nucleic acid. Preferably the hybridizationcomplex is immobilized. Either the target or the primer can be theimmobilized component. After annealing, the immobilized complexes arewashed to remove unbound nucleic acids. This is followed by an extensionreaction. This is the complexity reduction component of the assay.Subsequently, the extended probe is removed via the purification tag.The purified probe is then hybridized with allele specific probes (thespecificity component). The hybridized probes are then amplified (theamplification component).

In a preferred embodiment the allele specific probe contains universalpriming sites and an adapter sequence. Preferably the universal primingsites are specific for a particular allele. That is, one of theuniversal priming sites may be common to all alleles, but the seconduniversal priming site is specific for a particular allele. Followinghybridization the allele specific primer, the complexes are washed toremove unbound or mismatched primers. Thus, this configuration allowsfor allele specific amplification. Amplicons are detected as anindication of the presence of a particular allele.

In an alternative embodiment, the specificity component occurs first, Inthis embodiment allele specific probes are hybridized with the targetnucleic acid; an extension assay is performed whereby only the perfectlycomplementary probe is extended. That is, only the probe that isperfectly complementary to the probe at the interrogation positionserves as a substrate for extension reaction. Preferably the extensionreaction includes tagged, i.e. biotinylated, dNTPs such that theextension product is tagged. The extension product is then purified fromthe reaction mixture. Subsequently, a second allele specific primer ishybridized to the extension product. This step also serves as a secondspecificity step. In this embodiment the specificity steps also serve ascomplexity reduction components in that they enrich for target nucleicacids. Following the addition of the second allele specific primer andextension, the extension product is amplified, preferably with universalprimers.

As discussed previously, it is preferably for the at least one allelespecific primer to contain an allele specific priming site, preferablyan allele specific universal priming site. Again, this configurationallows for multiplexed allele specific amplification using universalprimers.

In an alternative embodiment, the target nucleic acid is firstimmobilized and hybridized with allele specific primers. Preferably theallele specific primers also include an adapter sequence that isindicative of the particular allele. Allele specific extension is thenperformed whereby only the primer that is perfectly complementary to thedetection position of the target nucleic acid will serve as a templatefor primer extension. That is, mismatched primers will not be extended.Of note, the allele specific position of the primer need not be the 3′terminal nucleotide of the primer. That is, the primer may extend beyondthe detection position of the target nucleic acid. In this embodiment itis preferable to include labeled dNTPs or ddNTPs or both such that theextension product is labeled and can be detected. In some preferredembodiments the interrogator is not the terminal position of the primer,but rather resides at a position 1, 2, 3, 4, 5 or 6 nucleotides from the3′ terminus of the primer.

In a preferred embodiment both dNTPs and ddNTPs are included in theextension reaction mixture. In this embodiment only one label is needed,and the amount of label can be determined and altered by varying therelative concentration of labeled and unlabeled dTNPs and ddNTPs. Thatis, in one embodiment labeled ddNTPs are included in the extension mixat a dilution such that each termination will result in placement ofsingle label on each strand. Thus, this method allows for quantificationof targets. Alternatively, if a higher signal is needed, a mixture oflabeled dNTPs can be used along with chain terminating nucleotides at alower concentration. The result is the incorporation of multiple labelsper extension product. Preferably the primers also include adapterswhich facilitate immobilization of the extension products for detection.

In an additional preferred configuration, target nucleic acids arehybridized with tagged locus specific primers. Preferably the primerincludes a locus specific portion and a universal priming site. Of note,as is generally true for locus specific primers, they need not beimmediately adjacent to the detection position. Upon hybridization, thehybridization complexes are immobilized, preferably by binding moietythat specifically binds the tag on the locus specific primer. Theimmobilized complexes are then washed to remove unlabeled nucleic acids;the remaining hybridization complexes are then subject to an extensionreaction. Following extension of the locus specific primer, a nucleotidecomplementary to the nucleotide at the detection position will beincorporated into the extension product. In some embodiments it isdesirable to limit the size of the extension because this reduces thecomplexity of subsequent annealing steps. This may be accomplished byincluding both dNTPs and ddNTPs in the reaction mixture.

Following the first extension, a second locus or allele specific primeris hybridized to the immobilized extension product and a secondextension reaction occurs. Preferably the second extension primerincludes a target specific portion and a universal priming site. Afterextension, universal amplification primers can be added to the reactionand the extension products amplified. The amplicons can then be used fordetection of the particular allele. This can be accomplished bycompetitive hybridization, as described herein. Alternatively, it can beaccomplished by an additional extension reaction. When the extensionreaction is performed, preferably a primer that contains an adaptersequence and a target specific portion is hybridized with the amplicons.Preferably the target specific portion hybridizes up to a position thatis adjacent to the detection position, i.e. the particular allele to bedetected. Polymerase and labeled ddNTPs are then added and the extensionreaction proceeds, whereby incorporation of a particular label isindicative of the nucleotide that is incorporated into the extensionprimer. This nucleotide is complementary to the nucleotide at thedetection position. Thus, analyzing or detecting which nucleotide isincorporated into the primer provides an indication of the nucleotide atthe allele position. The extended primer is detected by methods thatinclude but are not limited to the methods described herein.

In another embodiment, the genotyping specificity is conferred by theextension reaction. In this embodiment, two probes (sometimes referredto herein as “primers”) are hybridized non-contiguously to a targetsequence comprising, from 3′ to 5′, a first second and third targetdomain. Preferably the target is immobilized. That is, in a preferredembodiment, the target sequence is genomic DNA and is attached to asolid support as is generally described in U.S. Ser. No. 09/931,285,hereby expressly incorporated by reference in its entirety. In thisembodiment, magnetic beads, tubes or microtiter plates are particularlypreferred solid supports, although other solid supports as describedbelow can also be used.

The first probe hybridized to the first domain, contains a firstuniversal priming sequence and contains, at the 3′ end (within theterminal six bases), an interrogation position. In some preferredembodiments the interrogator is not the terminal position of the primer,but rather resides at a position 1, 2, 3, 4, 5 or 6 nucleotides from the3′ terminus of the primer. Subsequently, the unhybridized primers areremoved. This is followed by providing an extension enzyme such as apolymerase, and NTPs (which includes both dNTPs, NTPs and analogs, asoutlined below). If the interrogation position is perfectlycomplementary to the detection position of the target sequence, theextension enzyme will extend through the second target domain to form anextended first probe, ending at the beginning of the third domain, towhich the second probe is hybridized. A second probe is complementary tothe third target domain, and upon addition of a ligase, the extendedfirst probe will ligate to the second probe. The addition of a primerallows amplification to form amplicons. If the second probe comprises anantisense second primer, exponential amplification may occur, such as inPCR. Similarly, one or other of the probes may comprise an adapter oraddress sequence, which facilitates detection. For example, the adaptermay serve to allow hybridization to a “universal array”. Alternatively,the adapter may serve as a mobility modifier for electrophoresis or massspectrometry analysis, or as a label sequence for the attachment oflabels or beads for flow cytometry analysis.

In another embodiment, the reaction is similar except that it is theligation reaction that provides the detection position/interrogationspecificity. In this embodiment, it is the second probe that comprises a5′ interrogation position. The extended first probe will not be ligatedto the second probe if there is a mismatch between the interrogationposition and the target sequence. As above, the addition of a primerallows amplification to form amplicons. If the second probe comprises anantisense second primer, exponential amplification may occur, such as inPCR. Similarly, one or other of the probes may comprise an adapter oraddress sequence, which facilitates detection. For example, the adaptermay serve to allow hybridization to a “universal array”. Alternatively,the adapter may serve as a mobility modifier for electrophoresis or massspectrometry analysis, or as a label sequence for the attachment oflabels or beads for flow cytometry analysis.

Once prepared, and attached to a solid support as required, the targetsequence is used in genotyping or methylation detection reactions. Itshould be noted that while the discussion below focuses on certainassays, in general, for each reaction, each of these techniques may beused in a solution based assay, wherein the reaction is done in solutionand a reaction product is bound to the array for subsequent detection,or in solid phase assays, where the reaction occurs on the surface andis detected, either on the same surface or a different one.

The assay continues with the addition of a first probe. The first probecomprises, a 5′ first domain comprising a first universal primingsequence. The universal priming sites are used to amplify the modifiedprobes to form a plurality of amplicons that are then detected in avariety of ways, as outlined herein. In preferred embodiments, one ofthe universal priming sites is a T7 site, such that RNA is ultimatelymade to form the amplicon. Alternatively, as more fully outlined below,two universal priming sequences are used, one on the second probegenerally in antisense orientation, such that PCR reactions or otherexponential amplification reactions can be done. Alternatively, a singleuniversal primer can be used for amplification. Linear amplification canbe performed using the SPIA assay, T7 amplification, linear TMA and thelike, as described herein.

The first probe further comprises, 3′ to the priming sequence, a seconddomain comprising a sequence substantially complementary to the firsttarget domain of the target sequence. Again, the second target domaincomprises n nucleotides, wherein n is an integer of at least 1, andpreferably from 1 to 100 s, with from 1 to 10 being preferred and from1, 2, 3, 4 and 5 being particularly preferred. What is important is thatthe first and third target domains are non-contiguous, e.g. notadjacent.

In a preferred embodiment, the first probe, further comprises, 3′ to thesecond domain, an interrogation position within the 3′ six terminalbases. As used herein, the base which basepairs with a detectionposition base in a hybrid is termed a “readout position” or an“interrogation position”; thus one or the other of the first or secondprobes of the invention comprise an interrogation position, as outlinedherein. In some cases, when two SNP positions or detection positions arebeing elucidated, both the first and the second probes may compriseinterrogation positions.

When the first probe comprises the interrogation position, it fallswithin the six 3′ terminal nucleotides, with in three, and preferablytwo, and most preferably it is the 3′ terminal nucleotide. In somepreferred embodiments the interrogator is not the terminal position ofthe primer, but rather resides at a position 1, 2, 3, 4, 5 or 6nucleotides from the 3′ terminus of the primer. Alternatively, the firstprobe does not contain the interrogation position; rather the secondprobe does. This depends on whether the extension enzyme or the ligationenzyme is to confer the specificity required for the genotypingreaction.

In addition to the first probes of the invention, the compositions ofthe invention further comprise a second probe for each target sequence.The second probes each comprise a first domain comprising a sequencesubstantially complementary to the third target domain of a targetsequence as outlined herein.

In some embodiments, the second probes comprise a second universalpriming site. As outlined herein, the first and second probes cancomprise two universal primers, one in each orientation, for use in PCRreactions or other amplification reactions utilizing two primers. Thatis, as is known in the art, the orientation of primers is such to allowexponential amplification, such that the first universal primingsequence is in the “sense” orientation and the second universal primingsequence is in the “antisense” orientation.

In a preferred embodiment, it is the second probe that comprises theinterrogation position. In this embodiment, the second probe comprises a5′ interrogation nucleotide, although in some instances, depending onthe ligase, the interrogation nucleotide may be within 1-3 bases of the5′ terminus. However, it is preferred that the interrogation base be the5′ base.

In a preferred embodiment, either the first or second probe furthercomprises an adapter sequence, (sometimes referred to in the art as “zipcodes”) to allow the use of “universal arrays”. That is, arrays aregenerated that contain capture probes that are not target specific, butrather specific to individual artificial adapter sequences.

It should be noted that when two universal priming sequences and anadapter is used, the orientation of the construct should be such thatthe adapter gets amplified; that is, the two universal priming sequencesare generally at the termini of the amplification template, describedbelow.

The first and second probes are added to the target sequences to form afirst hybridization complexes. The first hybridization complexes arecontacted with a first universal primer that hybridizes to the firstuniversal priming sequence, an extension enzyme and dNTPs.

If it is the first probe that comprises the interrogation nucleotide, ofthe base at the interrogation position is perfectly complementary withthe base at the detection position, extension of the first primer occursthrough the second target domain, stopping at the 5′ of the secondprobe, to form extended first probes that are hybridized to the targetsequence, forming second hybridization complexes. If, however, the baseat the interrogation position is not perfectly complementary with thebase at the detection position, extension of the first probe will notoccur, and no subsequent amplification or detection will occur.

Extension of the enzyme will also occur if it is the second probe thatcomprises the interrogation position. Once extended, the extended firstprobe is adjacent to the 5′ end of the second probe. In the case wherethe interrogation position was in the first probe, the two ends of theprobes (the 3′ end of the first probe and the 5′ end of the secondprobe) are respectively perfectly complementary to the target sequenceat these positions, and the two probes can be ligated together with asuitable ligase to form amplification templates.

The conditions for carrying out the ligation will depend on theparticular ligase used and will generally follow the manufacturer'srecommendations.

If, however, it is the second probe that carries the interrogationposition at its 5′ end, the base at the interrogation position must beperfectly complementary to the detection position in the target sequenceto allow ligation. In the absence of perfect complementarity, nosignificant ligation will occur between the extended first probe and thesecond probe.

It should be noted that the enzymes may be added sequentially orsimultaneously. If the target sequences are attached to a solid support,washing steps may also be incorporated if required.

The ligation of the extended first probe and the second probe results inan amplification template comprising at least one, and preferably two,universal primers and an optional adapter. Amplification can then bedone, in a wide variety of ways. As will be appreciated by those in theart, there are a wide variety of suitable amplification techniquesrequiring either one or two primers, as is generally outlined in U.S.Ser. No. 09/517,945, now U.S. Pat. No. 6,355,431, hereby expresslyincorporated by reference.

Accordingly, the invention provides a method of identifying candidatedisease genes by identifying genes with altered methylation. That is,methylation patterns as detected by the methods described herein arecompared between healthy patients and sick or diseased patients.Alternatively, samples from healthy tissues are compared with samplesfrom sick or diseased tissues.

In addition the invention provides methods of diagnosing diseases. Thatis, as noted herein, certain aberrations in methylation patterns ofcertain genes results in diseases. According to the methods as describedherein these diseases can be diagnosed in a highly multiplex fashion. Inaddition, because the method also provides for identifying additionalmethylated genes or patterns, additional diseases related to aberrantmethylation of genes are diagnosed by the methods of the invention.

In the preferred method, the detection substrate used for any of theabove assays is a random array substrate, as described in U.S. Pat. No.6,023,540 which is incorporated by reference herein, where thehybridization of complementary nucleic acid sequences, or addresssequences, are used as the particular detection means. The arrays can bemanufactured with a standard set of nucleic acid address sequences, oneaddress sequence for each different nucleic acid to be detected. Thecomplementary nucleic acid sequences are provided as part of the linearnucleic acid sequences of the mutation-detection probes, inside of theworking portion of the amplification primers. During amplification, theaddress sequences are amplified along with each respectivemutation-detection probe. In order to detect the results of themultiplexed genotyping reaction, the resulting amplifiedmutation-detection probe mixture is applied to the array, whereby thecomplementary address sequences on the mutation-detection probes and onthe array hybridize, and the results are analyzed by known methods, suchas fluorescence.

Other detection schemes such as flow cytometry, mass spectroscopy,capillary electrophoresis, spotted arrays, or spatially-directed arrayscan also be used to simultaneously read the results of the multiplexednucleic acid detection reactions.

Accordingly, the present invention provides methods and compositionsuseful in the detection of nucleic acids, particularly the labeledamplicons outlined herein. As is more fully outlined below, preferredsystems of the invention work as follows. Amplicons are attached (viahybridization) to an array site. This attachment can be either directlyto a capture probe on the surface, through the use of adapters, orindirectly, using capture extender probes as outlined herein. In someembodiments, the target sequence itself comprises the labels.Alternatively, a label probe is then added, forming an assay complex.The attachment of the label probe may be direct (i.e. hybridization to aportion of the target sequence), or indirect (i.e. hybridization to anamplifier probe that hybridizes to the target sequence), with all therequired nucleic acids forming an assay complex.

Accordingly, the present invention provides array compositionscomprising at least a first substrate with a surface comprisingindividual sites. By “array” or “biochip” herein is meant a plurality ofnucleic acids in an array format; the size of the array will depend onthe composition and end use of the array. Nucleic acids arrays are knownin the art, and can be classified in a number of ways; both orderedarrays (e.g. the ability to resolve chemistries at discrete sites), andrandom arrays are included. Ordered arrays include, but are not limitedto, those made using photolithography techniques (Affymetrix GeneChip™),spotting techniques (Synteni and others), printing techniques (HewlettPackard and Rosetta), three dimensional “gel pad” arrays, etc. Apreferred embodiment utilizes microspheres on a variety of substratesincluding fiber optic bundles, as are outlined in PCTs US98/21193, nowpublished international application W099/18434, PCT US99/14387, nowpublished international application W099/67641 and PCT US 98/05025, nowpublished international application W099/67641; W098/50782; and U.S.Ser. No. 09/287,573, now U.S. Pat. Nos. 7,348,181, 09/151,877, now U.S.Pat. Nos.6,327,410, 09/256,943, now U.S. Pat. Nos. 6,429,027,09/316,154, now U.S. Pat. Nos. 6,364,790, 60/119,323, 09/315,584, nowU.S. Pat. No. 6,544,732; all of which are expressly incorporated byreference.

Arrays containing from about 2 different bioactive agents (e.g.different beads, when beads are used) to many millions can be made, withvery large arrays being possible. Generally, the array will comprisefrom two to as many as a billion or more, depending on the size of thebeads and the substrate, as well as the end use of the array, thus veryhigh density, high density, moderate density, low density and very lowdensity arrays may be made. Preferred ranges for very high densityarrays are from about 10,000,000 to about 2,000,000,000, with from about100,000,000 to about 1,000,000,000 being preferred (all numbers being insquare cm). High density arrays range about 100,000 to about 10,000,000,with from about 1,000,000 to about 5,000,000 being particularlypreferred. Moderate density arrays range from about 10,000 to about100,000 being particularly preferred, and from about 20,000 to about50,000 being especially preferred. Low density arrays are generally lessthan 10,000, with from about 1,000 to about 5,000 being preferred. Verylow density arrays are less than 1,000, with from about 10 to about 1000being preferred, and from about 100 to about 500 being particularlypreferred. In some embodiments, the compositions of the invention maynot be in array format; that is, for some embodiments, compositionscomprising a single bioactive agent may be made as well. In addition, insome arrays, multiple substrates may be used, either of different oridentical compositions. Thus for example, large arrays may comprise aplurality of smaller substrates.

In addition, one advantage of the present compositions is thatparticularly through the use of fiber optic technology, extremely highdensity arrays can be made. Thus for example, because beads of 200 μm orless (with beads of 200 nm possible) can be used, and very small fibersare known, it is possible to have as many as 40,000 or more (in someinstances, 1 million) different elements (e.g. fibers and beads) in a 1mm² fiber optic bundle, with densities of greater than 25,000,000individual beads and fibers (again, in some instances as many as 50-100million) per 0.5 cm² obtainable (4 million per square cm for 5μcenter-to-center and 100 million per square cm for 1μ center-to-center).

By “substrate”, “array substrate” or “solid support” or othergrammatical equivalents herein is meant any material that can bemodified to contain discrete individual sites appropriate for theattachment or association of beads and is amenable to at least onedetection method. It should be noted that the array substrate isdistinct from the “capture surface” described above. The capture surfaceis for the immobilization of target nucleic acids while the arraysubstrate is for detection of amplicons, i.e. the results of thedetection or genotyping assay. As will be appreciated by those in theart, the number of possible array substrates is very large. Possiblearray substrates include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, optical fiber bundles, and a variety of other polymers. Ingeneral, the array substrates allow optical detection and do notthemselves appreciably fluoresce.

Generally the array substrate is flat (planar), although as will beappreciated by those in the art, other configurations of substrates maybe used as well; for example, three dimensional configurations can beused, for example by embedding the beads in a porous block of plasticthat allows sample access to the beads and using a confocal microscopefor detection. Similarly, the beads may be placed on the inside surfaceof a tube, for flow-through sample analysis to minimize sample volume.Preferred substrates include optical fiber bundles as discussed below,and flat planar substrates such as paper, glass, polystyrene and otherplastics and acrylics.

In a preferred embodiment, the substrate is an optical fiber bundle orarray, as is generally described in U.S. Ser. No. 08/944,850, now U.S.Pat. No. 7,115,884, and Ser. No. 08/519,062, now U.S. Pat. No.6,200,737, PCT US98/05025, now published international applicationWO98/40726and PCT US98/09163, now published international applicationWO98/50782, all of which are expressly incorporated herein by reference.Preferred embodiments utilize preformed unitary fiber optic arrays. By“preformed unitary fiber optic array” herein is meant an array ofdiscrete individual fiber optic strands that are co-axially disposed andjoined along their lengths. The fiber strands are generally individuallyclad. However, one thing that distinguished a preformed unitary arrayfrom other fiber optic formats is that the fibers are not individuallyphysically manipulatable; that is, one strand generally cannot bephysically separated at any point along its length from another fiberstrand.

Generally, the array of array compositions of the invention can beconfigured in several ways; see for example U.S. Ser. No. 09/473,904,now U.S. Pat. No. 6,858,394,hereby expressly incorporated by reference.In a preferred embodiment, as is more fully outlined below, a “onecomponent” system is used. That is, a first substrate comprising aplurality of assay locations (sometimes also referred to herein as“assay wells”), such as a microtiter plate, is configured such that eachassay location contains an individual array. That is, the assay locationand the array location are the same. For example, the plastic materialof the microtiter plate can be formed to contain a plurality of “beadwells” in the bottom of each of the assay wells. Beads containing thecapture probes of the invention can then be loaded into the bead wellsin each assay location as is more fully described below. Arrays aredescribed in U.S. Pat. No. 6,023,540 and U.S. Ser. No. 09/151,877, filedSep. 11, 1998, now U.S. Pat. No. 6,327,410, Ser. No. 09/450,829, filedNov. 29, 1999, now U.S. Pat. No. 6,266,459, Ser. No. 09/816,651, filedMar. 23, 2001, now abandoned, and Ser. No. 09/840,012, filed Apr. 20,2001, now abandoned, all of which are expressly incorporated herein byreference. In addition, other arrays are described in 60/181,631, filedFeb. 10, 2000, Ser. No. 09/782,588, filed Feb. 12, 2001, now abandoned,Ser. No. 60/113,968, filed Dec. 28, 1998, Ser. No. 09/256,943, filedFeb. 24, 1999, now U.S. Pat. No. 6,429,027 Ser. No. 09/473,904, filedDec. 28, 1999, now U.S. Pat. No. 6,858,394 and Ser. No. 09/606,369,filed Jun. 28, 2000, now Abandoned, all of which are expresslyincorporated herein by reference.

Alternatively, a “two component” system can be used. In this embodiment,the individual arrays are formed on a second substrate, which then canbe fitted or “dipped” into the first microtiter plate substrate. Apreferred embodiment utilizes fiber optic bundles as the individualarrays, generally with “bead wells” etched into one surface of eachindividual fiber, such that the beads containing the capture probes areloaded onto the end of the fiber optic bundle. The composite array thuscomprises a number of individual arrays that are configured to fitwithin the wells of a microtiter plate.

By “composite array” or “combination array” or grammatical equivalentsherein is meant a plurality of individual arrays, as outlined above.Generally the number of individual arrays is set by the size of themicrotiter plate used; thus, 96 well, 384 well and 1536 well microtiterplates utilize composite arrays comprising 96, 384 and 1536 individualarrays, although as will be appreciated by those in the art, not eachmicrotiter well need contain an individual array. It should be notedthat the composite arrays can comprise individual arrays that areidentical, similar or different. That is, in some embodiments, it may bedesirable to do the same 2,000 assays on 96 different samples;alternatively, doing 192,000 experiments on the same sample (i.e. thesame sample in each of the 96 wells) may be desirable. Alternatively,each row or column of the composite array could be the same, forredundancy/quality control. As will be appreciated by those in the art,there are a variety of ways to configure the system. In addition, therandom nature of the arrays may mean that the same population of beadsmay be added to two different surfaces, resulting in substantiallysimilar but perhaps not identical arrays.

At least one surface of the substrate is modified to contain discrete,individual sites for later association of microspheres. These sites maycomprise physically altered sites, i.e. physical configurations such aswells or small depressions in the substrate that can retain the beads,such that a microsphere can rest in the well, or the use of other forces(magnetic or compressive), or chemically altered or active sites, suchas chemically functionalized sites, electrostatically altered sites,hydrophobically/hydrophilically functionalized sites, spots of adhesive,etc.

The sites may be a pattern, i.e. a regular design or configuration, orrandomly distributed. A preferred embodiment utilizes a regular patternof sites such that the sites may be addressed in the X-Y coordinateplane. “Pattern” in this sense includes a repeating unit cell,preferably one that allows a high density of beads on the substrate.However, it should be noted that these sites may not be discrete sites.That is, it is possible to use a uniform surface of adhesive or chemicalfunctionalities, for example, that allows the attachment of beads at anyposition. That is, the surface of the substrate is modified to allowattachment of the microspheres at individual sites, whether or not thosesites are contiguous or non-contiguous with other sites. Thus, thesurface of the substrate may be modified such that discrete sites areformed that can only have a single associated bead, or alternatively,the surface of the substrate is modified and beads may go down anywhere,but they end up at discrete sites. That is, while beads need not occupyeach site on the array, no more than one bead occupies each site.

In a preferred embodiment, the surface of the substrate is modified tocontain wells, i.e. depressions in the surface of the substrate. Thismay be done as is generally known in the art using a variety oftechniques, including, but not limited to, photolithography, stampingtechniques, molding techniques and microetching techniques. As will beappreciated by those in the art, the technique used will depend on thecomposition and shape of the substrate.

In a preferred embodiment, physical alterations are made in a surface ofthe substrate to produce the sites. In a preferred embodiment, thesubstrate is a fiber optic bundle and the surface of the substrate is aterminal end of the fiber bundle, as is generally described in Ser. No.08/818,199 and Ser. No. 09/151,877, both of which are hereby expresslyincorporated by reference. In this embodiment, wells are made in aterminal or distal end of a fiber optic bundle comprising individualfibers. In this embodiment, the cores of the individual fibers areetched, with respect to the cladding, such that small wells ordepressions are formed at one end of the fibers. The required depth ofthe wells will depend on the size of the beads to be added to the wells.

Generally in this embodiment, the microspheres are non-covalentlyassociated in the wells, although the wells may additionally bechemically functionalized as is generally described below, cross-linkingagents may be used, or a physical barrier may be used, i.e. a film ormembrane over the beads.

In a preferred embodiment, the surface of the substrate is modified tocontain chemically modified sites, that can be used to attach, eithercovalently or non-covalently, the microspheres of the invention to thediscrete sites or locations on the substrate. “Chemically modifiedsites” in this context includes, but is not limited to, the addition ofa pattern of chemical functional groups including amino groups, carboxygroups, oxo groups and thiol groups, that can be used to covalentlyattach microspheres, which generally also contain corresponding reactivefunctional groups; the addition of a pattern of adhesive that can beused to bind the microspheres (either by prior chemicalfunctionalization for the addition of the adhesive or direct addition ofthe adhesive); the addition of a pattern of charged groups (similar tothe chemical functionalities) for the electrostatic attachment of themicrospheres, i.e. when the microspheres comprise charged groupsopposite to the sites; the addition of a pattern of chemical functionalgroups that renders the sites differentially hydrophobic or hydrophilic,such that the addition of similarly hydrophobic or hydrophilicmicrospheres under suitable experimental conditions will result inassociation of the microspheres to the sites on the basis ofhydroaffinity. For example, the use of hydrophobic sites withhydrophobic beads, in an aqueous system, drives the association of thebeads preferentially onto the sites. As outlined above, “pattern” inthis sense includes the use of a uniform treatment of the surface toallow attachment of the beads at discrete sites, as well as treatment ofthe surface resulting in discrete sites. As will be appreciated by thosein the art, this may be accomplished in a variety of ways.

In some embodiments, the beads are not associated with a substrate. Thatis, the beads are in solution or are not distributed on a patternedsubstrate.

In a preferred embodiment, the compositions of the invention furthercomprise a population of microspheres. By “population” herein is meant aplurality of beads as outlined above for arrays. Within the populationare separate subpopulations, which can be a single microsphere ormultiple identical microspheres. That is, in some embodiments, as ismore fully outlined below, the array may contain only a single bead foreach capture probe; preferred embodiments utilize a plurality of beadsof each type.

By “microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. The composition of the beadswill vary, depending on the class of capture probe and the method ofsynthesis. Suitable bead compositions include those used in peptide,nucleic acid and organic moiety synthesis, including, but not limitedto, plastics, ceramics, glass, polystyrene, methylstyrene, acrylicpolymers, paramagnetic materials, thoria sol, carbon graphite, titaniumdioxide, latex or cross-linked dextrans such as Sepharose, cellulose,nylon, cross-linked micelles and Teflon may all be used. “MicrosphereDetection Guide” from Bangs Laboratories, Fishers Ind. is a helpfulguide.

The beads need not be spherical; irregular particles may be used. Inaddition, the beads may be porous, thus increasing the surface area ofthe bead available for either capture probe attachment or tagattachment. The bead sizes range from nanometers, i.e. 100 nm, tomillimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200microns being preferred, and from about 0.5 to about 5 micron beingparticularly preferred, although in some embodiments smaller beads maybe used.

Each microsphere comprises a capture probe, although as will beappreciated by those in the art, there may be some microspheres which donot contain a capture probe, depending on the synthetic methods.

Attachment of the nucleic acids may be done in a variety of ways, aswill be appreciated by those in the art, including, but not limited to,chemical or affinity capture (for example, including the incorporationof derivatized nucleotides such as AminoLink or biotinylated nucleotidesthat can then be used to attach the nucleic acid to a surface, as wellas affinity capture by hybridization), cross-linking, and electrostaticattachment, etc. In a preferred embodiment, affinity capture is used toattach the nucleic acids to the beads. For example, nucleic acids can bederivatized, for example with one member of a binding pair, and thebeads derivatized with the other member of a binding pair. Suitablebinding pairs are as described herein for IBL/DBL pairs. For example,the nucleic acids may be biotinylated (for example using enzymaticincorporate of biotinylated nucleotides, for by photoactivatedcross-linking of biotin). Biotinylated nucleic acids can then becaptured on streptavidin-coated beads, as is known in the art.Similarly, other hapten-receptor combinations can be used, such asdigoxigenin and anti-digoxigenin antibodies. Alternatively, chemicalgroups can be added in the form of derivatized nucleotides, that canthem be used to add the nucleic acid to the surface.

Similarly, affinity capture utilizing hybridization can be used toattach nucleic acids to beads. Alternatively, chemical crosslinking maybe done, for example by photoactivated crosslinking of thymidine toreactive groups, as is known in the art.

In a preferred embodiment, each bead comprises a single type of captureprobe, although a plurality of individual capture probes are preferablyattached to each bead. Similarly, preferred embodiments utilize morethan one microsphere containing a unique capture probe; that is, thereis redundancy built into the system by the use of subpopulations ofmicrospheres, each microsphere in the subpopulation containing the samecapture probe.

As will be appreciated by those in the art, the capture probes mayeither be synthesized directly on the beads, or they may be made andthen attached after synthesis. In a preferred embodiment, linkers areused to attach the capture probes to the beads, to allow both goodattachment, sufficient flexibility to allow good interaction with thetarget molecule, and to avoid undesirable binding reactions.

In a preferred embodiment, the capture probes are synthesized directlyon the beads. As is known in the art, many classes of chemical compoundsare currently synthesized on solid supports, such as peptides, organicmoieties, and nucleic acids. It is a relatively straightforward matterto adjust the current synthetic techniques to use beads.

In a preferred embodiment, the capture probes are synthesized first, andthen covalently attached to the beads. As will be appreciated by thosein the art, this will be done depending on the composition of thecapture probes and the beads. The functionalization of solid supportsurfaces such as certain polymers with chemically reactive groups suchas thiols, amines, carboxyls, etc. is generally known in the art.Accordingly, “blank” microspheres may be used that have surfacechemistries that facilitate the attachment of the desired functionalityby the user. Some examples of these surface chemistries for blankmicrospheres include, but are not limited to, amino groups includingaliphatic and aromatic amines, carboxylic acids, aldehydes, amides,chloromethyl groups, hydrazide, hydroxyl groups, sulfonates andsulfates.

When random arrays or liquid arrays are used, an encoding/decodingsystem must be used. For example, when microsphere arrays are used, thebeads are generally put onto the substrate randomly; as such there areseveral ways to correlate the functionality on the bead with itslocation, including the incorporation of unique optical signatures,generally fluorescent dyes, that could be used to identify the nucleicacid on any particular bead. This allows the synthesis of the captureprobes to be divorced from their placement on an array, i.e. the captureprobes may be synthesized on the beads, and then the beads are randomlydistributed on a patterned surface. Since the beads are first coded withan optical signature, this means that the array can later be “decoded”,i.e. after the array is made, a correlation of the location of anindividual site on the array with the bead or probe at that particularsite can be made. This means that the beads may be randomly distributedon the array, a fast and inexpensive process as compared to either thein situ synthesis or spotting techniques of the prior art.

When liquid arrays are used, beads to which the amplicons areimmobilized can be analyzed by FACS. Again, beads can be decoded todetermine which amplicon is immobilized on the bead. This is anindication of the presence of the target analyte.

However, the drawback to these methods is that for a large array, thesystem requires a large number of different optical signatures, whichmay be difficult or time-consuming to utilize. Accordingly, methods foranalysis and decoding of arrays are described in Ser. No. 08/944,850,filed Oct. 6, 1997, now U.S. Pat. No. 7,115,884, PCT/US98/21193, filedOct. 6, 1998, now published international application WO99/18434, Ser.No. 09/287,573, filed Apr. 6, 1999, now U.S. Pat. No. 7,348,181,PCT/US00/09183, filed May 6, 2000, now published internationalapplication WO00/60332, Ser. No. 60/238,866, filed Oct. 6, 2000, Ser.No. 60/119,323, filed Feb. 9, 1999, Ser. No. 09/500,555, filed Feb. 9,2000, now abandoned, Ser. No. 09/636,387, filed Aug. 9, 2000, nowAbandoned, Ser. No. 60/151,483, filed Aug. 30, 1999, Ser. No.60/151,668, filed Aug. 31, 1999, Ser. No. 09/651,181, filed Aug. 30,2000, now U.S. Pat. No. 6,942,948, Ser. No. 60/272,803, filed Mar. 1,2001, all of which are expressly incorporated herein by reference. Inaddition, methods of decoding arrays are described in 60/090,473, filedJun. 24, 1998, Ser. No. 09/189,543, filed Nov. 10, 1998, now abandoned,Ser. No. 09/344,526, filed Jun. 24, 1999, now U.S. Pat. No.7,060,431PCT/US99/14387, filed Jun. 24, 1999, now publishedinternational application WO99/67641, Ser. No. 60/172,106, filed Dec.23, 1999, Ser. No. 60/235,531, filed Sep. 26, 2000, Ser. No. 09/748,706,filed Dec. 22, 2000, now U.S. Pat. No. 7,033,754, and provisionalapplication entitled Decoding of Array Sensors with Microspheres, filedJun. 28, 2001 (no serial number received), all of which are expresslyincorporated herein by reference.

As outlined herein, the present invention finds use in a wide variety ofapplications. All references cited herein are incorporated by reference.

EXAMPLES Example 1 Attachment of Genomic DNA to a Solid Support

1. Fragmentation of Genomic DNA

Human Genomic DNA 10 _g (100 μl) 10× DNase I Buffer 12.5 μl DNase I (1U/_μl, BRL) 0.5 μl ddH2O 12 μlIncubate 37° C. for 10 min. Add 1.25 μl 0.5 M EDTA, Heat at 99° C. for15 min.2. Precipitation of Fragmented Genomic DNA

DNase I fragmented genomic DNA 125 μl Quick-Precip Plus Solution (EdgeBiosystems)  20 μl Cold 100% EtOH 300 μlStore at −20° C. for 20 min. Spin at 12,500 rpm for 5 min. Wash pellet2× with 70% EtOH, and air dry.3. Terminal Transferase End-Labeling with Biotin

DNase I fragmented and precipitated genomic DNA (in H2O) 77.3 μl 5×Terminal transferase buffer 20 μl Biotin-N6-ddATP (1 mM, NEN) 1 μlTerminal transferase (15 U/μl) 1.7 μl37° C. for 60 min. Add 1 μl 0.5 M EDTA, then heat at 99° C. for 15 min4. Precipitation of Biotin-Labeled Genomic DNA

Biotin-labeled genomic DNA 100 μl Quick-Precip Solution  20 μl EtOH 250μl−20° C. for 20 min and spin at 12,500 rpm for 5 min, wash 2× with 70%EtOH and air dry.5. Immobilization of Biotin-Labeled Genomic DNA to Streptavidin-CoatedPCR TubesHeat-Denature Genomic DNA for 10 min on 95° C. Heat Block.

Biotin-labeled genomic DNA (0.3 g/μl)  3 μl _× binding buffer 25 μl SNPPrimers (50 nM) 10 μl ddH2O 12 μlIncubate at 60° C. for 60 min.

-   Wash 1× with 1× binding buffer,-   1× with 1× washing buffer,-   1× with 1× ligation buffer.-   1× binding buffer: 20 mM Tris-HCl, pH7.5, 0.5M NaCl, 1 mM EDTA, 0.1%    SDS.-   1× washing buffer: 20 mM Tris-HCl pH7.5, 0.1 M NaCl, 1 mM EDTA, 0.1%    Triton X-100.-   1× ligation buffer: 20 mM Tris-HCl pH7.6, 25 mM Potassium acetate,    10 mM magnesium acetate, 10 mM DTT, 1 mM NAD, 0.1% Triton X-100.    6. Ligation in Streptavidin-Coated PCR Tubes-   make a master solution and each tube contains 49 μl 1× ligation    buffer-   μl Taq DNA Ligase (40 U/_μl)-   incubate at 60° C. for 60 min.-   wash each tube 1× with 1× washing buffer-   1× with ddH2O    7. Elution of Ligated Products-   add 50 μl ddH2O to each tube and incubated at 95° C. for 5 min,    chilled on ice, transfer the supernatant to a clean tube.    8. PCR Set Up

25 mM dNTPs 0.5 μl 10× buffer II (PEB) 2.5 μl 25 mM MgCl2 1.5 μlAmpliTaq Gold DNA Polymerase (5 Units/μl, PEB) 0.3 μl Eluted (ligated)product (see above) 3 μl Primer set (T3/T7/T7v, 10 _M each) 2 μl ddH2O 1μl Total volume 25 μlPCR condition:

-   94° C. 10 min-   35 cycles of 94° C. 30 sec-   60° C. 30 sec    and then-   72° C. 30 sec

Example 2 Methylation Detection Assays

Plasmid DNA was used as an independent control DNA. The quality ofmethylation was tested by restriction digest of umnethylated andmethylated DNA by methylation sensitive enzyme Hpa II and itsisoschisomer Msp I, which is not sensitive to methylation. Bands werenot detected on an agarose gel after digestion with methylated pUC19with Hpa II for two hours at 37° C., while the unmethylated DNA wascompletely digested (data not shown).

A set of OLA primers targeted to 5 different Hpa II sites on thepBluescript KS+ plasmid were designed. Four out of these 5 sites arealso present on pUC19. The specificity of these primers was tested in amodel experiment using a standard genotyping protocol on the bead array.Methylated or unmethylated plasmid DNA was spiked into a human genomicDNA sample at approximately 1:1 molar ratio (1 pg of plasmid DNA to 1 ugof human genomic DNA). Samples were digested by restriction enzymes DraI or Dra I in combination with methylation sensitive Hpa II. (FIG. 13a). This experiment confirmed that we could distinguish unmethylated andmethylated control DNAs using restriction digestion. The primers did notcrossreact with human genomic DNA and can be used in variouscombinations with other gene-specific primers (FIG. 13 b).

Example 3 Whole Genome Amplification of Bi-Sulfite Converted Genomic DNA

Using standard DNA polymerase, this approach takes advantage of theunique sequence feature of genomic DNAs after bisulfite treatment, i.e.all the un-methylated cytosines are converted to uracil. Therefore, theDNA template for amplification will only contain three bases, A, G andT, and will be single-stranded, as opposed to the regular gDNA template.

Genomic amplification can be performed with a mixture of two sets ofprimers that contain all possible combinations of three nucleotides, inparticular a first set having A, T and C, but not G; and a second sethaving A, T and G, but not C. Primers from the first set will havehigher affinity to the original bisulfite converted DNA strand, whileprimers from the second set will preferentially anneal to the newlysynthesized complementary strand. This approach avoids the presence of Gand C in the same primer, thus preventing the primers to cross over anyCpG sites to be interrogated, but nevertheless allows both strands to beamplified. In a variation of this gene amplification method, a poly-Aprimer is used for the first strand synthesis in combination withprimers, containing combinations of A, T and G, but not C, for thecomplementary strand. This variation avoids the bias that may beintroduced by the un-balanced annealing efficiency of primerscorresponding to the two alleles (C or T).

Alternatively, the genomic amplification can be performed with primersequences that contain only Adenines (As). The homopoly-A primers, forexample, 6-mer, 9-mer, or longer, can be used for the first strandsynthesis. After that, a homopoly-T tail can be added to the 3′-ends ofthe first strand products, using terminal deoxyribonucleotidetransferase (TdT). A standard PCR is used to amplify the bisulfiteconverted genomic DNAs using a poly-A primers. It was estimated that thepoly-T frequencies in the genome after bisulfite conversion are, onaverage, such that the physical distance between any two poly-(T)nsequences (n>=9) is 330 bp, which is a convenient amplicon size range.

Briefly, for the sodium bisulfite reaction, reagents should be freshprepared before use.

To prepare 2 M NaOH 400 mg NaOH pellets are dissolved in 5 ml H₂O. A 2.5M metabisulfite solution (corresponding to a free bisulfiteconcentration of 5M as follows: 1.9 g of sodium metabisulfite (Na₂S₂O₄,Sigma) are mixed with 2 ml sterile H₂O and 0.7 ml 2M NaOH and themixture is shaken for 10-15 minutes. Simultaneously, 350 mg ofhydroquinone are mixed with 1 ml sterile H₂O to yield a saturatedsolution. Subsequently, 500 ul of hydroquinone solution are added to thebisulfite solution and shaking of the mixture is continued. The pH ischecked using indicator paper and adjusted, if necessary, to pH 5.0 with2M NaOH. The final volume is adjusted to 4 ml using sterile H₂O (˜200ul).

Samples of 100 ul final volume, 3 M Bisulfite are prepared by startingwith a 36 ul sample (˜1 ug GDNA) and 4 ul 2N NaOH, incubating for 10 minat 37° C. and adding 60 ul 5M Bisulfite solution. The samples aresubjected to thermocycling under the following conditions:

One cycle at 95° C. 90 sec, 50° C. for 1 hour; four cyles at 95° C. for45 sec followed by 50° C. for 4 hours. The samples are then stored at 4°C.

To finish the bisulfite modifications a MultiScreen-PCR clean-up plateis used to clean the reactions from Bisulfite. The samples aretransferred into the plate wells; and unused wells are covered withadhesive seal. The plates are placed on top of the MultiScreen manifoldand a vacuum (manometer reads 15) is applied for 10 min or until wellshave emptied. The filters appear shiny even after they are dry. Thereaction mixtures are washed with 100 ul of ddH₂O, followed by additionof 50 ul of water to each well and vigorous mixing of the samples on aplate shaker (1750 rpm) for 5 minutes. The eluted DNA is transferredfrom each well into an Eppendorf tube followed by addition of 50 ul of0.4 M NaOH to a final concentration of 0.2 M. The eluted DNA isincubated at RT for 20 min to desulfonate the pyrimidines followed byaddition of ½ volume (50 ul) of 7.5 M ammonium acetate and 2.5 vol (375ul) of 100% Ethanol. After centrifugation for 10 min at 12000 g, thepellets are washed with 70% EtOH, air-dried, and resuspended in 20 ul TEfor biotinylation.

Example 4 Genome-Wide Calibration of Genomic DNA Methylation Measurement

For any given organism, a reference genomic DNA is amplified many fold,for example, 100 fold, 1000 fold or more. Any whole genome amplificationmethod desired by the user can be utilized, for example, randomhexamers, 9-mers, 11-mers, 13-mers or 15-mers, primed DNA amplificationby enzymes including, but are not limited to, the Klenow fragment of DNApolymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNApolymerase and Phi29 DNA polymerase or any other DNA polymerase, orRubicon Genomics' OmniPlex technology. After this amplification, all theendogenous DNA methylations are diluted 100-fold, 1000-fold or more. Theamplified genomic DNA serves as a fully “un-methylated” template. Theamplified genomic DNA or the original un-amplified genomic DNA, or bothare subsequently methylated in vitro, and then serve as a fully“methylated” template.

The fully un-methylated and methylated genomic DNA templates can bemixed at different ratios, for example, 0%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% and, 100% of methylated template, to constitute agradient of methylated template concentration. Methylation assaysperformed on these mixed templates will allow for generation of acalibration curve for the quantitative methylation measurement inunknown samples. The mixed templates can also be used to determine theLimit of Detection (LOD) for any methylation detection method, inparticular what percentage of the methylated template can be detected inthe presence of un-methylated template.

This approach can also be used to evaluate the integrity of amethylation assay across the entire genome and thus facilitate thedevelopment of further methylation assays. For example, if an assayresults in identical methylation measurements for both the un-methylatedand the methylated templates, the integrity of the assay has beencompromised.

Example 5 Genomic Amplification by Sodium Bisulfite Reaction

Briefly, for the sodium bisulfite reaction, reagents should be freshprepared before use.

To prepare 2 M NaOH, 400 mg NaOH pellets are dissolved in 5 ml H2O.Subsequently, prepare a 2.5 M metabisulfite solution corresponding to afree bisulfite concentration of 5M as follows by mixing 1.9 g of sodiummetabisulfite (Na₂S₂O₄, Sigina) with 2 ml sterile H₂O and 0.7 ml 2MNaOH. The miture is shaken for 10-15 minutes. Simultaneously, 350 mg ofhydroquinone are mixed with 1 ml sterile H₂O to achieve a saturatedsolution. Then, 500 ul of hydroquinone solution are added to thebisulfite solution and shaking is continued. The pH is checked usingindicator paper and adjusted, if necessary, to pH 5.0 with 2M NaOH. Thefinal volume is adjusted to 4 ml using sterile H2O (˜200 ul).

Samples of 100 ul final volume, 3 M Bisulfite are prepared by startingwith a 36 ul sample (˜1 ug gDNA) and 4 ul 2N NaOH, incubating for 10 minat 37° C. and adding 60 ul 5M Bisulfite solution. The samples aresubjected to thermocycling under the following conditions:

One cycle at 95° C. 60 sec, 50° C. for 1 hour; four cycles at 95° C.each for 20 sec followed by 50° C. for 4 hours. The samples are thenstored at 4° C.

To finish the bisulfite modifications a Micro-PCR clean-up plate andin-plate desulfonation are used to clean the reactions from Bisulfite.The samples are transferred into the plate wells; and unused wells arecovered with adhesive seal. The plates are placed on top of theMillipore manifold and a vacuum (manometer reads 20) is applied for 8minutes or until wells have emptied. The filters appear shiny even afterthey are dry. The reaction mixtures are washed with 100 ul of ddH₂O for5 to 6 minutes. Subsequently, 20 ul of 0.1 N NaOH are added to each welland the samples are mixed vigorously on a plate shaker (1750 rpm) for 10minutes, followed by addition of 10 ul of 7.5 M ammonium acetate toneutralize samples in the plate.

A vacuum is applied for 6-8 min or until wells have emptied. The samplesare subsequently washed with 100 ul of 10 mM Tris-HCl pH 8.0 and elutedin 20 ul TE-buffer on the shaker, followed by recovery of the elutedDNA.

All references are expressly incorporated herein by reference.

1. A method for multiplex detection of methylation in a population ofdouble-stranded target nucleic acids comprising: (a) providing apopulation of double-stranded target nucleic acids labeled with apurification tag, wherein said target nucleic acids comprise potentiallymethylated target sequences; (b) cleaving said population of targetnucleic acids with an enzyme, whereby said enzyme selectively cleaves atunmethylated target sequences in said population of target nucleic acidsand does not cleave at methylated target sequences in said population oftarget nucleic acids, forming a population of cleaved target nucleicacids labeled with a purification tag and a population of non-cleavedtarget nucleic acids labeled with a purification tag; (c) immobilizingsaid population of non-cleaved said target nucleic acids by saidpurification tag, thereby forming immobilized non-cleaved target nucleicacids; and (d) detecting the presence of said immobilized non-cleavedtarget nucleic acids wherein said detecting step comprises: contactingsaid immobilized non-cleaved target nucleic acids with a compositioncomprising a plurality of different target probes, each of said probescomprising: a first region complementary to a first region of one ofsaid immobilized non-cleaved target nucleic acids and a second regioncomprising a detection sequence complementary to one of said potentiallymethylated target sequences, whereby selectively forming a plurality ofdifferent hybridization complexes between said immobilized non-cleavedtarget nucleic acids and said different target probes indicates thepresence of methylation in said population of double-stranded targetnucleic acids.
 2. The method according to claim 1, wherein saidpurification tag comprises biotin.
 3. The method according to claim 1,wherein said enzyme is HpaII.
 4. A method for multiplex detection ofmethylation in a population of double-stranded target nucleic acidscomprising: (a) providing a population of double-stranded target nucleicacids labeled with a purification tag, wherein said target nucleic acidscomprise potentially methylated target sequences; (b) cleaving saidpopulation of target nucleic acids with an enzyme, whereby said enzymeselectively cleaves at unmethylated target sequences in said populationof target nucleic acids and does not cleave at methylated targetsequences in said population of target nucleic acids, forming apopulation of cleaved target nucleic acids labeled with a purificationtag and a population of non-cleaved target nucleic acids labeled with apurification tag; (c) immobilizing said population of non-cleaved saidtarget nucleic acids by said purification tag, thereby formingimmobilized non-cleaved target nucleic acids; and (d) detecting thepresence of said immobilized non-cleaved target nucleic acids, whereinsaid detecting step comprises: (i) contacting said immobilizednon-cleaved target nucleic acids with a composition comprising aplurality of different target probes and selectively forming a pluralityof different hybridization complexes between said immobilizednon-cleaved target nucleic acids and said different target probes, eachof said probes comprising: a region complementary to a region of one ofthe non-cleaved target nucleic acid and at least a universal primingsequence; (ii) amplifying said different hybridization complexes in thepresence of a composition comprising: a) at least first universalprimers complementary to said universal priming sequence; b) dNTPs; andc) a polymerase, thereby forming a plurality of different amplicons,whereby detecting said plurality of different amplicons is an indicationof the presence of methylation in said population of double-strandedtarget nucleic acids.
 5. The method according to claim 4, wherein saidpurification tag comprises biotin.
 6. The method according to claim 4,wherein said enzyme is HpaII.